DANNY VIJEY JEYARAJU

La protease rhomboide PARL, nouveau contrôleur de l'apoptose et de la régulation dela morphologie mitochondriale:

découverte des mécanismes moléculaires responsables de son activité

Mémoire présenté

à la Faculté des études supérieures de l'Université Laval

dans le cadre du programme de maîtrise en neurobiologie

pour l'obtention du grade de maître es sciences (M.Sc.)

FACULTÉ DE MÉDECINE

UNIVERSITÉ LAVAL

QUÉBEC

2007

© Danny Vijey Jeyaraju, 2007

Abstract

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner

and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the

rnolecular mechanism behind mitochondrial morphology has been recruited to govern novel

functions, such as development, calcium signaling, and apoptosis, which suggests that novel

mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Hère we

show that phosphorylation and cleavage of the vertebrate-specifïc PP domain of the mammalian

presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology.

Phosphorylation of three residues embeddedin this domain, Ser-65, Thr-69, and Ser-70, impair a

cleavage at position Ser77|Ala78 that is required to initiate PARL-induced mitochondrial

fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact

mitochondrial dynamics, providing a blueprint to study the molecular évolution of

mitochondrial morphology.

Résumé

Le remodelage de la morphologie et de la structure de des cristae des mitochondries est un

processus dynamique régulé par un ensemble conservé de protéines qui coordonnent la « fusion »

et la « fission » de la membrane interne et externe de l'organelle. Pendant l'évolution des

métazoaires, les mécanismes moléculaires qui contrôlent ces processus ont été recrutés pour

régler de nouvelles fonctions, tels le développement, la signalisation du calcium, et l'apoptose.

Ceci suggère donc que des mécanismes, toujours inconnus, doivent exister pour réguler la

machinerie de la fusion/fission des membranes mitochondriales. Dans cette étude nous

démontrons que la phosphorylation et le clivage d'un domaine de la protéase rhomboide PARL

(presenilin-associated rhomboid-like), lequel est présent uniquement chez les vertébrés règlent la

morphologie des mitochondries. Nous montrons que la phosphorylation de trois acides aminés

conservés dans ce domaine, la Ser-65, la Thr-69 et la Ser-70, empêche un clivage aux positions

Ser77-Ala78 qui est requis pour initier la fragmentation mitochondriale induite par PARL. Nos

résultats démontrent que la phosphorylation et le clivage de PARL ont un impact sur la

dynamique des mitochondries, ce qui nous fournit un modèle pour étudier l'évolution

moléculaire de la morphologie des mitochondries.

Avant-propos

Authors contributions in the accompanying paper entitled "Phosphorylation and cleavage ofpresenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrialmorphology" by Jeyaraju DV, Xu L, Letellier MC, Bandaru S, Zunino R, Berg EA, McBrideHM, and Pellegrini L.

Danny Jevaraju:

1. Performed ail the experiments in Figs. 1, 4, and 5C;

2. Generated the PARL mutants listed in the following page;

3. Purified (/) endogenous PARL from mitochondria isolated from human placenta, (//) wildtype PARL and mutant STS 65-69-70 AAA, from transfected HeLa cells (for subséquentmasspec analysis);

4. Developed and characterized the anti PARL-C-Term antibody for immunoprecipitationand western blots analysis.

Liqun Xu;

1. Performed the experiments in Fig. 3 and 5A.

2. Isolated mitochondria from human placenta;

3. Donated her placenta.

Marie Claude Letellier:

1. Generated ail the PARL A mutants shown in Fig 4B, and the STS 65-69-70 AAA andSTS 65-69-70 DDD shown in Fig 4A;

2. Isolated MAMP from HEK293 (for subséquent masspec analysis).

Sirisha Bandaru and Rodolfo Zunino:

Provided technical assistance in the Pellegrini and McBride labs, respectively.

Dr. Eric Berg:

Performed the mass spectrometric analysis shown in Fig. 2.

Dr Heidi McBride:

1. Directed and supervised the experiments shown in Figs. 3, 5A and 5B;

2. Co-wrote the manuscript.

Dr. Luca Pellegrini:

1. Conceived, initiated, and directed the study;

2. Designed the experiments;

3. Wrote the manuscript.

Avant-propos (continued)

List ofPARL mutants and constructs generated by Danny Jeyaraju and used in this study

STS 65-69-70 AAD

STS 65-69-70 ADA

STS 65-69-70 ADD

STS 65-69-70 DAA

STS 65-69-70 DAD

STS 65-69-70 DDA

A 84-87 + STS 65-69-70 DDD

Wild type PARL Myc-CT

S277G Myc-CT

STS 65-69-70 AAA Myc-CT

STS 65-69-70 DDD Myc-CT

A 84-87 Myc-CT

A 87-90 Myc-CT

Acknowledgements

On the outset, I would like to thank my parents Dr. M. A. Jeyaraju and Mrs. T. Punithavathy and

my sister Dr. J. Pamela for providing me the means and encouragement to corne out of my

country and pursue my dreams in a scientific career. They had been always supportive of me

through times thick and thin. They always had let me do what I loved to do and without their

support and love I wouldn't hâve been able to achieve this in my life and I dedicate this work to

them.

I come from India, a country with strong traditional and cultural values. In my culture, respect is

an important virtue and there are three great personalities to respect. First your Parents, second

your Mentor, and third God Almighty. Dr Luca Pellegrini, fits perfectly in the définition of

Mentor in my culture, which means more than just being the boss. He was very kind and

generous in helping me with the adaptation process in a new country with a new language and

new culture. He helped me right from buying clothes to facilitating me to meet top notch

scientists. I thank him profoundly for giving wings to my dreams and for a scintillating start to

my scientific career with a PNAS paper. He helped realize my scientific potential and bring it out

in the best possible way. I could keep going on to say about him but I would leave the rest for my

PhD thesis!

I take this opportunity to thank my professors in India, Dr M. Patrick Gomez, a great human

being who inspired me ail through my studies in India, and Dr Gabriel Melchias, a perfectionist

who was a rôle model for me in my académie formation, both are from St.Joseph's Collège,

Tiruchirappalli, India and they provided me a perfect springboard to launch my career.

Sincère thanks to Dr Heidi McBride for the sharing of her expertise which was very important in

the success of the PNAS paper. I also would like to thank her for the trust she has always shown

towards me.

I thank the Almighty for showering me with blessings, guiding me ail the way and making it

possible for me to live my dreams and my life.

I thank ail my friends who hâve taken care of my parents while I am at the other end of the world.

Spécial words of thanks go to Shemy Mary Louis and Marie Stella, India, who had given me the

impetus to come out of the country and pursue my dreams.

Heartfelt and sincère thanks go to David Schaendel and Eugénie Legratiet, France, for having

helped me corne out of the adaptation stress. They had been instrumental in the beginning of a

social life for me right from taking me out and in teaching me french and making me the best

man in their wedding. I also would like to thank my friends in Québec City Vicky Jobin, Barbara

Gravel, Laurea Rock, Gaston and Gael Coté, Jasmin Matthew, Stéphanie Jobin, Estefania

Echeverry and the friendly guys in the gas station around the corner of my street whose name I

never knew. Ail thèse people had contributed in signifïcant ways in my adaptation hère.

Big thanks to Katalin Tôth, Emma and Leonardo for being caring and loving towards me. They

provided me with a home away from home and I thank them sincerely for that.

Spécial word of thanks goes to Paula Power, Newfoundland for helping me get through times

when I was down.

I would like to thank my friends Chafford Luçon Alfred, Asif Hussain, Linda Suzanne David,

Pradeep Devakumar, Joseph Devadoss, Prasanna, Prasad, Kabilan, N. Rajkumar, Thirupathy,

Gopi, Rajkumar, Hari, Sreeram, Robert, Sekar, Ajay Vasanth, Jaisankar Rajappan, Edward,

Sasikumar, Abinandhan, Sundar, Renuka Ramaraja, Rowena Maninang and Patricia for ail the

love and wishes I got from them.

I would like to thank Simon Labrecque, Hugues Dufour and Nathalie Lavoie for helping me with

french.

I would like to thank ail the people I hâve known in my beautiful country, India and hère in La

belle province Québec who hâve ail contributed in some way, known or unknown to make me

what I am today.

Table of Contents

Abbreviations Page 8

1 Introduction Page 9

I. Mitochondrial Rhomboids: regulators of mitochondria morphology Page 9remodelling

> Rhomboids: enzymatic activity, évolution, and structure

> Mitochondrial rhomboids: novel components of the mitochondria morphologyremodelling machinery

> The mammalian mitochondrial rhomboid protease PARL

II. Régulation of apoptosis by mitochondria morphology Page 13

> Apoptosis: rôle of cytochrome c in the intrinsic pathway> The mitochondria morphology machinery

> Mechanisms of mitochondrial morphology régulation integrated to the apoptotic program

> Rôle of the mitochondrial rhomboid protease PARL in mitochondria morphology andapoptosis

^ Molecular mechanisms regulating PARL activity

2 Objectives Page 20

3 Results and Discussion Page 20

> Reprint of Jeyaraju et al., PNAS (2006)

4 Perspectives Page 21

5 Materials and methods Page 24

6 Bibliography Page 38

Abbreviations

PARL - Presenilin associated rhomboid like protein

MAMP - Mature mitochondrial protein

PACT - PARL C-terminal product

PDB - Protein Data Bank

Pcpl - Processing of cytochrome c peroxidase protein 1

GST - Glutathione S transferase

ATP - Adenosine triphosphate

0PA1 - Optic Atrophia 1

GTP - Guanosine triphosphate

DRP1 - Dynamin-related protein 1

hFis - human Mitochondrial fission 1 protein

Rbdlp - Rhomboid protein 1

IMS - Intermembrane Space

PKA - Protein Kinase A

PINK1 - PTEN-induced putative kinase protein 1

GSK - Glycogen synthase kinase

CK - Casein Kinase

MtDNA - Mitochondrial DNA

ROS - Reactive Oxygen Species

Rtg - Rétrograde régulation protein

TCA - Tricarboxylic Acid

CCCP - Carbonyl cyanide m-chlorophenylhydrazone

Introduction

1. Mitochondrial Rhomboids: regulators of mitochondria morphology remodelling

Rhomboids: enzymatic activity, évolution, and structure

Regulated intramembrane proteolysis (RIP), is a new paradigm of signal transduction, which

appears to be prominent in ail forms of life (Brown et al 2000). Under RIP, membrane-bound

proteins undergo site-specific proteolysis within one of their transmembrane hélices (TMH). This

processing releases a moiety of the protein that, through intracellular relocation or extracellular

release, typically exécutes the signalling fonction of the membrane-tethered precursor protein.

This mechanism influences processes as diverse as lipid metabolism, cellular differentiation, the

response to unfolded proteins, mitochondria morphology, and apoptosis.

Enzymatically, RIP requires proteases that, despite the water-excluding environment of the lipid

bilayer, somehow are able to hydrolyze their transmembrane substrates. This processing is thus

executed by a very unique class of proteases, named I-Clips, for intramembrane-cleaving

proteases (Weihofen & Martoglio 2003). To date, only three distinct I-Clip families hâve been

discovered. The first family, whose prototypic member is the human site-two protease (S2P) that

cleaves and activâtes sterol regulatory élément binding proteins (SREBPs), are a group of

metalloproteases (Brown & Goldstein 1999, Rawson et al 1997). A second family, whose

prototypic members are the presenilins (PSs) involved in cleavage of the amyloid-B precursor

protein (ApPP) and Notch, are a group of aspartic proteases (De Strooper et al 1999, De Strooper

et al 1998). This family also includes the signal peptide peptidase (SPP) (Weihofen et al 2002),

which catalyses intramembrane proteolysis of signal séquence remnants and possibly also

membrane proteins in the endoplasmic reticulum (ER) membrane of animal and plant cells

(Weihofen & Martoglio 2003). The third and most recently discovered I-Clip family are the

rhomboids (Urban'2006), which are the most evolutionary conserved one and, by implication, the

first to émerge in life (Koonin et al 2003).

The conserved core of rhomboid family members consists of six conserved TMHs (Koonin et al

2003), with the Ser and His residues required to form the catalytic dyad (Urban & Wolfe 2005)

embedded in TMH-4 and TMH-6, respectively (Wang et al 2006). Consistent with the widely

accepted hypothesis that I-Clips catalytic centre is assembled within the plane of the membrane,

récent crystallographic studies hâve shown that rhomboid's catalytic dyad is found at the protein

9

E.xUac»lluhit

A A r)irr/ Cyloptasm

Notch

M

interior at a depth below the membrane surface, indicating that, in RIP, the scission of the peptide

bonds indeed takes place within the hydrophobic environment of the membrane bilayer (Wang et

al 2006).

Fig 1.1 Représentation of the four known

families of intramembrane proteases. Catalytic

residues are in red, and conserved motifs that

are typical of proteases of each mechanistic

class are in black. Note that presenilins and

Rhomboids cleave only type I proteins (with an

extracellular/luminal N terminus), whereas site

2 proteases and signal-peptide peptidases

cleave type II tproteins. The approximate sites

of cleavage are shown, and the green arrow

indicates the direction of domain release: only

Rhomboids are involved predominantly in

extracellular release of factors. Presenilins and

signal-peptide peptidases are aspartyl

proteases that use two aspartates to cleave substrates, the site 2 proteases are metalloproteases that coordinate a

zinc ion using two conserved htstidines and an aspartate, and Rhomboids are serine proteases that use a catalytic

triad to cleave substrates (hydrogen bonds of the triad are indicated as dashed lines). Taken from Urban, S., and

Freeman, M. (2002) Intramembrane proteolysis controls diverse signalling pathways throughout évolution. Curr Opin

Genêt Dev 12, 512-518

À -*^fl

In spite of the présence of rhomboids in the majority of modem life forms from ail three primary

superkingdoms, phylogenetic analysis suggests that this family has not been inherited from the

last universal common ancestor (LUCA) (Koonin et al 2003). Instead, the tree topology indicates

that this family emerged in some bacterial lineage and afterwards had been widely disseminated

by horizontal gène transfer (HGT), and then lost in some lineages. Both archaea and eukaryotes

hâve acquired rhomboids on several independent occasions. In particular, at least two HGT

events hâve contributed to the origin of eukaryotic rhomboids, one of them yielding the RHO

subfamily and the other one the PARL subfamily, with a possible additional HGT in plants

(Koonin et al 2003), where rhomboids could regulate plastids morphology and activities.

Eukaryotic members of the two subfamilies hâve différent domain architecture organization.

Proteins of the RHO subfamily, which is prototyped by the drosophila developmental regulator

Rhomboid (Bier et al 1990), typically hâve an extra TMH added carboxy-terminally to the 6-

10

TMH catalytic core, the "6+1" structure. Instead, members of the PARL subfamily, which is

prototyped by the mitochondrial morphology regulator Presenilin-associated rhomboid-like

protein (PARL) (Pellegrini et al 2001), hâve an extra TMH added to the amino terminus of the 6-

TMH catalytic core, the "1+6" structure (Koonin et al 2003). Such additional TMH in either

subfamily implies the existence of a loop that connects it to the 6-TMH catalytic core, which

could hâve a regulatory function.

TM6

Figure 1.2 Structures of GlpG rhomboid

intramembrane serine proteases from £.

coli from différent laboratories. AH

structures reveal six transmembrane hélices

and a Ser201-His254 catalytic dyad located

near the N termini of TM4 and TM6,

respectively. The short TM4 and TM5 hélices

(~15 residues) aid the formation of a central

water-filled cavity containing the catalytic

dyad, which is located about 10 A below the

extracellular surface of the membrane bilayer.

The water molécules in the cavity, shown as

van der Waals sphères, are based on Wang et

al. (Wang et al 2006) (purple) and Wu. et al.

(Wu et al 2006)(green). Thèse structures (PDB

2IC8, 2NRF and 2IRV) crystallized in the P32,

P3i and P2i space groups with one, two

(antiparallel) and two (antiparallel) molécules

in the asymmetric unit, respectively. Each of

the antiparallel molécules in the 2NRF and 2IRV asymmetric units hâve slightly différent structures, (a) View of GlpG

structures from the extracellular surface, based on Wu et al., showing the alignment of molécules A and B of 2NRF

(2.6 A, molécule A red, molécule B yellow) relative to the single molécule of 2IC8 based an Wang et al., (2.1 A, grey).

Ail parts of the three molécules align very well, except for TM5. In chain A, this hélix is distorted and displaced away

from the central cavity, compared with TM5 of 2IC8. In chain B, TM5 is also displaced, but less than in chain A. Loop

L5 blocks access to the cavity in 2IC8 (gray) but not in chain A of 2NRF (red). (b) View of GlpG structures in a

direction parallel to the membrane plane. This view toward the catalytic dyad reveals a direct pathway to the dyad via

a TM5-TM2 'gâte', (c) View, perpendicular to the membrane, of structures in a, with aligned structures of Ben-Shem et

al (Ben-Shem) (2.3 A, 21RV, molécule A cyan, molécule B pink) added. TM5 is displaced away from the cavity in both

molécules A and B, but not as much as in 2NRF. (d) View as in b of the five aligned structures. The great variability of

TM5 in the structures suggests that TM5 is easily distorted in the crystal structures, consistent with TM5 being easily

displaced in vivo to allow entry of the substrate hélix. Taken from Stephen H White (2006) Rhomboid intramembrane

protease structures galore! Nature Structural & Molecular Biology -13, 1049 -1051

I I

Mitochondrial rhomboids: novel components of the mitochondria morphology remodelling

machinery

A yeast genetic screen aimed at identifying components involved in mitochondrial fusion led to

the identification of two mutants, Ugo 1 and Ugo 2, which were defective in this process (Sesaki

& Jensen 2001). Ugo 2 was later described as a mutant of Pcpl (Esser et al 2002, Sesaki et al

2003), a gène encoding a rhomboid protease of the PARL subfamily which is targeted to the

mitochondria in vitro (Steinmetz et al 2002) and in vivo (Esser et al 2002). Récent évidences hâve

shown that mitochondrial rhomboids participate in the régulation of the organelle's morphology.

Several groups hâve indeed shown that yeast cells lacking the yeast rhomboid Pcplp are

defective in the processing of cytochrome c peroxidase 1 (Ccpl) (Esser et al 2002, McQuibban et

al 2003) and of the dynamin-related Mgml protein (Herlan et al 2003, McQuibban et al 2003,

Sesaki et al 2003), a key component of the mitochondria fusion machinery (Guan et al 1993,

Shepard & Yaffe 1999, Wong et al 2000). While the fonction of Ccpl cleavage is still unknown,

that of Mgmlp is linked to mitochondria morphology régulation (Herlan et al 2003, McQuibban

et al 2003). Consistently, APcpl cells contain partially fragmented mitochondria, instead of the

long tubular branched mitochondria of wild-type cells (Dimmer et al 2002, Sesaki & Jensen

2001, Sesaki et al 2003). However, this phenotype is rescued by expression of the drosophila and

mammalian ortholog of Pcplp, Rhomboid-7 and PARL, respectively (McQuibban et al 2003).

Thus, during animal évolution the function of this rhomboid protease in the régulation of

mitochondrial morphology has been conserved, which is reflected by their localization in the

inner mitochondrial membrane (Jeyaraju et al 2006) (Dimmer et al 2002) and identical "1+6 "

structure, with the catalytic serine and histidine located in TMH5 and TMH7, respectively

(Koonin et al 2003).

The mammalian mitochondrial rhomboid protease PARL

PARL, presenilin-associated rhomboid-like, dérives its name from being identified in a yeast two

hybrid screen for proteins that could interact with Alzheimer's presenilin protein (Pellegrini et al

2001). Although Presenilin-1 has been found in the mitochondria (Ankarcrona & Hultenby 2002,

Gupta et al 2004), its association in vivo with PARL remains to be addressed. The first functions

of a PARL protease were inferred from a discovery in the budding yeast: early genome-wide

screens revealed that a homolog of PARL, Pcpl (also called Rbdl and YgrlOlw), is localized to

12

mitochondria (Steinmetz et al 2002), and its deletion results in mitochondrial fragmentation

(Dimmer et al 2002). Pcpl was subsequently discovered to be responsible for cleaving the

targeting séquence of cytochrome c peroxidase (Ccpl), a nuclear-encoded mitochondrial protein

présent in the intermembrane space (Esser et al 2002). This defined a fourth and previously

unknown targeting peptide processing pathway in mitochondria. However, pcpl deleted cells had

a slow growth phenotype while the ccpl deletion did not, suggesting that the basis of the pcpl

phenotype results from failure to process another substrate.

Despite the functional and structural conservation of yeast Pcplp, drosophila Rhomboid-7, and

mammalian PARL, their N-terminal domain are unrelated (Sik et al 2004). The N-terminal région

of PARL, which is in the matrix (Jeyaraju et al 2006), spans the first 100 amino acids of the

protein and shows no détectable similarity to any other available protein séquences. This domain

of PARL, designated Pp domain, is vertebrate-specifïc, as indicated by the notable conservation

among mammals and, to a lesser extent, other vertebrates, but not between vertebrates and insects

(Sik et al 2004). Although the function of the Pp domain remains unknown, its biological

relevance is évident from its séquence conservation. Indeed, in the four available mammalian

PARL séquences, 58 of the 62 residues of the Pp domain are invariant, and there are no insertions

or deletions, which suggests that at least during mammalian évolution, the N-terminal région of

PARL was subject to strong purifying sélection, which can be explained by functional

constraints. In unconstrained séquences evolving neutrally, very few, if any, invariant residues

would be expected to survive the -100 million years of évolution separating mammalian orders.

This analysis suggests that émergence of the Pp domain at the outset of vertebrate évolution may

be associated with the appearance of a new mechanism of régulation of PARL in mitochondria

morphology and/or with the recruitment of PARL into novel mitochondrial pathways.

II. Régulation of apoptosis by mitochondria morphology Apoptosis

Programmed cell death and its morphologie manifestation of apoptosis is a conserved pathway

that in its basic tenets appears operative in ail metazoans. Cell deaths during embryonic

development are essential for successful organogenesis and the crafting of complex multicellular

tissues. Apoptosis also opérâtes in adult organisms to maintain normal cellular homeostasis. This

is especially critical in long-lived mammals that must integrate multiple physiological as well as

pathological death signais, which for example includes regulating the response to infectious

13

agents. Gain- and loss-of-function models of gènes in the core apoptotic pathway indicate that the

violation of cellular homeostasis can be a primary pathogenic event that results in disease.

Evidence indicates that insufficient apoptosis can manifest as cancer or autoimmunity, while

accelerated cell death is évident in acute and chronic degenerative diseases, immunodeficiency,

and infertility.

Apoptosis: rôle of cytochrome c in the intrinsic pathway

Apoptosis can be triggered through two major pathways. Extracellular signais such as members

of the tumour necrosis factor family can activate the receptor-mediated extrinsic pathway.

Alternatively, stress signais such as DNA damage, hypoxia, and loss of survival signais may

trigger the mitochondrial intrinsic pathway. Within this pathway, in response to apoptotic stimuli,

cytochrome c, a previously known component of électron transfer chain in mitochondria, gets

released to cytosol where it binds to a partner protein, Apaf-1 (Liu et al 1996, Shi 2002). Apaf-1

consists of three functional domains; the N-terminal caspase recruitment domain (CARD), the

middle nucleotide-binding and oligomerization domain, and the C-terminal regulatory région

composed of 13 WD-40 repeats (Zou et al 1999). This regulatory région normally keeps Apaf-1

in an autoinhibitory state and when cytochrome c binds to this région, Apaf-1 becomes activated

in the présence of dATP or ATP (Acehan et al 2002, Adrain et al 1999, Srinivasula et al 1998).

The activation is accomplished through oligomerization of seven individual Apaf-1/cytochrome c

complexes into a wheel-like heptamer, called apoptosome (Acehan et al 2002, Zou et al 1999).

Once bound to the apoptosome, caspase-9 is activated, and subsequently triggers a cascade of

effector caspases activation and proteolysis, leading to apoptotic cell death.

Under non-apoptotic conditions, cytochrome c is kept confined inside "cisternae", which are

formed by juxtaposition of the cristae (Mannella 2006a, Mannella 2006b). Therefore, a crucial

step in the apoptotic program is to remodel the cristae to open thèse cisternae and liberate

cytochrome c into the intermembrane space (IMS) (Scorrano et al 2002), from where it can be

released to the cystosol through permeabilization of the outer membrane (Forte & Bernardi 2006,

Zamzami & Kroemer 2003). Conversely, under steady-state conditions the integrity of the

cisternae is essential to maintain cell viability (Scorrano et al 2002). Although récent work

suggested that the BH3-only pro-apoptotic Bcl-2 member Bid, disrupts the structure of the

dynamin-related protein OPA1, which is involved in keeping the cristae closed under steady-state

14

conditions, the only molécules known to maintain cristae closed under steady-state conditions are

the mitochondrial rhomboid protease PARL and the dynamin-related GTPase OPAl (Cipolat et

al 2006, Frezza et al 2006).

The mitochondria morphology machinery

Mitochondria form a functional reticulum whose steady-state morphology is regulated by

dynamic membrane fission and fusion events (Shaw & Nunnari 2002, Yaffe 2003). Typically,

suppression of the molecular mechanisms coordinating mitochondria membrane fusion, or

dominance of those governing membrane fission, cause mitochondria to fragment into short rods

or sphères. Conversely, disruption of the molecular processes regulating mitochondria membrane

fission, or prevalence of those controlling membrane fusion, generate elongated, interconnected

Mitochondrial shape is déterminée! by the antagonistic forcesof fission and fusion.

Stîmulated fission

Arrested fusionmFused Wild type Fragmented

tubules.

Fig 1.3. The différent states of mitochondria morphology.

Pioneering work in Saccharomyces cerevisiae has shown that mitochondria morphology is

governed by a small but growing set of conserved "mitochondria-shaping" proteins that

independently regulate membrane fusion and fission (Meeusen & Nunnari 2005, Okamoto &

Shaw 2005, Yaffe 2003). In the last years, some of their mammalian orthologues hâve been

identified, although their mechanism of régulation is still unexplored (Chen & Chan 2005).

15

In mammals, mitochondrial fission relies on hFis (Koch et al 2005, Mozdy et al 2000, Yoon et al

2003) and on a member of the dynamin family of GTPases, DRP1 (Kuroiwa et al 2006).

Mechanistically, it has been suggested that DRP1, like other dynamins (Schrader 2006),

oligomerizes into ring-like structures around the fission sites, to constrict the organelle in the

points where it divides (Ingerman et al 2005).

Mammalian mitochondrial fusion relies on yet three other GTPases from the dynamin family.

Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) are integrated within the mitochondrial outer

membrane, with their GTPase and coiled-coil domains exposed to the cytosol (Chan et al 2006,

Chen & Chan 2005, Chen et al 2005, Chen et al 2003). MFN1 and MFN2 exist as homotypic and

heterotypic complexes that can form between adjacent organelles. Mechanistically, it has been

suggested that the carboxy-terminal coiled coils tether two organelles undergoing fusion, with the

GTPase domains probably regulating the fusion reaction (Koshiba et al 2004).

Another dynamin-like GTPase, OPA1 (Alexander et al 2000, Delettre et al 2000), résides in the

intermembrane space, where it is associated with the inner membrane (Olichon et al 2002). OPA1

exists as multiple splice and cleavage variants, which ultimately control its activity in membrane

fusion (Delettre et al 2001, Frezza et al 2006, Ishihara et al 2006). Cleavage of OPA1 and of its

yeast orthologue Mgmlp is mediated by the rhomboid protease PARL, which therefore is also

part of the machinery regulating mitochondria morphology remodelling (Cipolat et al 2006,

Herlan et al 2004, Herlan et al 2003, McQuibban et al 2003). The mechanism controlling OPA1

processing is still unknown.

Mechanisms of mitochondrial morphology régulation integrated to the apoptotic program

It has been long known that in mammalian cells mitochondria undergo fragmentation during

apoptosis. However, only recently this process was shown to be an intégral part of the apoptotic

program, and not an epiphenomenon. In a pionering study by Frank, Youle and colleagues, this

process was found to be inhibited by ectopic expression of the dominant-négative mutant of the

pro-fission protein DRP1, DRP1-K38A (Frank et al 2001). More importantly, DRP1-K38A also

severely reduced the extent of cell death by delaying cytochrome c release, indicating that

apoptosis initiation requires mitochondria fragmentation. Since this pivotai observation, during

the past 5 years more évidence emerged indicating that the machinery governing the coordinated

fusion and fission of the outer and inner mitochondrial membrane participâtes in the progression

16

of apoptosis (Karbowski & Youle 2003, Youle & Karbowski 2005). For instance, in mammalian

cells down-regulation of the pro-fission protein hFisl powerfully inhibits cell death to an extent

signifïcantly greater than down-regulation of DRPl and at a stage of apoptosis distinct from that

induced by DRPl inhibition (Alirol et al 2006, Yu et al 2005). In addition, it has been shown that

cells depleted of the inner membrane pro-fusion protein OPA1 are extremely sensitive to

exogenous apoptosis induction, indicating that OPA1 may normally fonction as an antiapoptotic

protein (Lee et al 2004b). Finally, the outer membrane pro-fusion proteins MFN1 and MFN2,

alone or in combination, also prevent death induced by several intrinsic stimuli (Cipolat et al

2004, Neuspiel et al 2005, Sugioka et al 2004), consistent with early inhibition of MFN1-

dependent fusion during apoptosis (Karbowski et al 2004).

Although it is now well established that "mitochondria-shaping" proteins can positively and

negatively regulate apoptosis, little is known about how their coordinated activity is integrated

into the apoptotic program (Chan et al 2006), thereby limiting our understanding on whether

altered régulation of thèse proteins participâtes in tumour initiation and anti-cancer drug

résistance.

Rôle of the mitoehondrial rhomboid protease PARL in mitochondria morphology andapoptosis

Pioneering studies of mitoehondrial structure by Mannella and Frey using électron tomography

hâve shown that the well-known lamellar and tubular structure of the mitoehondrial inner

membrane générâtes a novel type of compartment, created by juxtaposition of inner-membrane

folds (Frey & Mannella 2000). Thèse intm-cristae structures form cisternae in which cytochrome

c is trapped (Mannella 2006a, Mannella 2006b), thereby forming a barrier to the diffusion of the

protein to the IMS (Frey & Mannella 2000). A critical step is thus required for apoptosis

progression: the opening of the cristae to eliminate the diffusion barrier and free cytochrome c to

the IMS, from where it can be released to the cytosol (Scorrano et al 2002).

Dr Pellegrini's lab has recently contributed to show that this process is mediated by the rhomboid

intramembrane-cleaving protease PARL (Cipolat et al 2006), a conserved regulator of

mitochondria morphology remodeling (McQuibban et al 2003). Mechanistically, PARL

coordinates the intramembraneous cleavage of the inner membrane protein OPA1, a single-pass

transmembrane GTPase, to liberate an IMS-soluble form of the protein (IMS-OPA1) that

assembles in macromolecular complexes with PARL and with the uncleaved, membrane bound

17

form of OPA1. This structure "staple" cristae juxtaposition (Cipolat et al 2006, Frezza et al

2006), thereby precluding cytochrome c access to the IMS. Consistent with this finding, we

showed that lack of PARL blocked IMS-OPA1 formation, causing faster cristae remodelling,

cytochrome c mobilization in the IMS, and apoptosis progression. Conversely, increased IMS-

OPA1 expression in the IMS protected MEF cells from several intrinsic pro-apoptotic stimuli

(Cipolat et al 2006). Thus, by maintaining cristae juxtaposed PARL "keeps a lid to apoptosis"

(Zhang et al 2003). Whether in some cancer cells PARL activity is upregulated is not known,

although this may be a mechanism tumour cells develop to acquire insensitivity to intrinsic

proapoptotic stimuli or résistance to anti-cancer drugs.

Although the exact components and stoichiometry of the molécules forming the macromolecular

complex that staple cristae juxtaposition are still unknown, évidence indicates the présence of at

least two uncleaved, membrane-bound forms of OPA1 and one IMS-OPA1. Given the fact that

OPA1 and PARL directly interact together via the first IMS loop of PARL, a model has been

proposed where two molécules of PARL on opposite sides of the cristae in juxtaposition each

bind a molécule of OPA1 which, in turn, binds to one molécule of IMS-OPA1 (Cipolat et al

2006, Frezza et al 2006). The mechanism that "unstaples" cristae juxtaposition during apoptosis

and release the PARL/OPA1/IMS-OPA1 complex is unknown. In addition, whether and how

increased IMS-OPA1 expression, which protects cells from apoptosis (Cipolat et al 2006),

stabilizes the PARL/OPA1/IMS-OPA1 complex remain undiscovered.

Molecular mechanisms regulating PARL activity

Despite their functional and structural conservation, the yeast mitochondrial rhomboid protease

Pcplp and PARL hâve unrelated N-terminal domains. The N-terminal région of PARL shows no

détectable similarity to any other available protein séquences. This région of PARL, designated

PP (spanning amino acids 40-100), is vertebrate-specifïc, as indicated by the notable

conservation among mammals and, to a lesser extent, other vertebrates, but not between

vertebrates and insects (11). Although the function of the P(3 domain remains unknown, its

biological relevance is évident from its séquence conservation. Indeed, in the four available

mammalian PARL séquences, 58 of the 62 residues of the Pp domain are invariant, and there are

no insertions or deletions (11), which suggests that at least during mammalian évolution, the N-

terminal région of PARL was subject to strong purifying sélection, which can be explained by

is

functional constraints. In unconstrained séquences evolving neutrally, very few, if any, invariant

residues would be expected to survive the 100 million years of évolution separating mammalian

orders (12, 13). This analysis suggests that émergence of the Pp domain at the outset of

vertebrate évolution may be associated with the appearance of a new mechanism of régulation of

PARL. We hâve recently shown that this part of the PARL molécule undergoes two consécutive

cleavage events, termed a and p. The proximal a-cleavage is a constitutive processing associated

with the protein import in the mitochondria, whereas the distal p-cleavage is regulated through a

mechanism of proteolysis requiring PARL activity supplied in trans (11). Whether this cleavage

occurs in vivo is unknown. In addition, its mechanism of régulation and its functional

significance remain unexplored.

19

Objectives

Dr. Pellegrini has recently shown that The PB domain of PARL undergoes to cleavage at position

76S|A77. This cleavage through a mechanism of proteolysis requiring PARL activity supplied in

trans. Whether this cleavage occurs in vivo is unknown. In addition, its mechanism of régulation

and its functional significance remain unexplored. Therefore, the goals of this study are to:

1 ) Investigate whether PARL B-cleavage occurs in vivo;

2) Discover the mechanism of régulation of PARL B-cleavage;

3) Address the rôle of PARL B-cleavage in mitochondria dynamics.

Results

Jeyaraju DV, Xu L, Letellier MC, Bandaru S, Zunino R, Berg EA, McBride HM, Pellegrini L.

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes

changes in mitochondrial morphology.

Proc Natl AcadSci USA 2006 Dec 5; 103(49): 18562-7

20

Phosphorylation and cleavage of presenilin-associatedrhomboid-like protein (PARL) promûtes changes inmitochondrial morphologyDanny V. Jeyaraju*, Liqun Xu\ Marie-Claude Letellier*, Sirisha Bandaru*, Rodolfo Zunino1, Eric A. Berg*,Heidi M. McBridet§, and Luca Pellegrini*6

'Centre de Recherche Université Laval Robert-Giffard, 2601 ch. de la Canardière, Québec City, QC, Canada G1J 2G3; fUniversity of Ottawa Heart Institute,40 Ruskin Street, Ottawa, ON, Canada K1Y4W7; and *21st Century Biochemicals, 33 Locke Drive, Marlboro, MA 01752-1146

Edited by Walter Neupert, Institute fur Physiologische Chemie, Munich, Germany, and accepted by the Editorial Board October 12, 2006 (received for reviewJune 14, 2006)

Remodeling of mitochondria is a dynamic process coordinated byfusion and fission of the inner and outer membranes of the organelle,mediated by a set of conserved proteins. In metazoans, the molecularmechanism behind mitochondrial morphology has been recruited togovorn novel functions, such as development, calcium signaling, andapoptosis, which suggests that novel mechanlsms should exist toregulate the conserved membrane fusion/fission machinery. Hère weshow that phosphorylation and cleavage of the vertebrate-specif ic P/3domain of the mammalian presenilin-associated rhomboid-like(PARL) protease can influence mitochondrial morphology. Phosphor-ylation of three residues embedded in this domain, Ser-65, Thr-69, andSer-70, impair a cleavage at position Ser77-Ala78 that is required toinitiate PARL-induced mitochondrial fragmentation. Our findings re-veal that PARL phosphorylation and cleavage impact mitochondrialdyniimics, providing a blueprint to study the molecular évolution ofmitochondrial morphology.

protein évolution | protein phosphorylation | rhomboids | mitochondrialdynamics | intramebrane proteolysis

M itochondrial biogenesis is an essential cellular processgoverned by a small set of proteins with membrane

pro-fusion and pro-fission activities which are conserved in aileukaryotes (1-3). During metazoan évolution, this process hasbeen recruited to coordinate novel mitochondrial functions, suchas apoptosis (4-6), thereby suggesting the émergence, in highereukaryotes, of novel mechanisms of régulation of the fusion andfission machinery of the organelle. Formai, mechanistic évi-dence supporting this hypothesis is, however, still missing.

Recently, rhomboid proteases hâve been implicated in the rég-ulation of mitochondrial membrane remodeling. Studies in Sac-charomyces cerevisiae demonstrated that PCP1P is required tocleave Mgmlp, an intermembrane space dynamin family memberthat participâtes in mitochondrial fusion events (7, 8). The yeastPCP1P protein belongs to a subfamily of mitochondrial rhomboidproteases typified by presenilin-associated rhomboid-like (PARL)protein (9, 10), the human ortholog of PCP1P (8). Despite theirfunctional and structural conservation, PCP1P and PARL hâveunrelated N-terminal domains. The N-terminal région of PARLshows no détectable similarity to any other available proteinséquences. This région of PARL, designated P/3 (spanning aminoacids 40-100), is vertebrate-specific, as indicated by the notableconservation among mammals and, to a lesser extent, other verté-brales, but not between vertebrates and insects (11). Although thefunction of the P)3 domain remains unknown, its biological rele-vance is évident from its séquence conservation. Indeed, in the fouravailable mammalian PARL séquences, 58 of the 62 residues of theP/3 domain are invariant, and there are no insertions or deletions(11), which suggests that at least during mammalian évolution, theN-terminal région of PARL was subject to strong purifying sélec-tion, which can be explained by functional conslraints. In uncon-strained séquences evolving neutrally, very few, if any, invariant

residues would be expected to survive the «=100 million years ofévolution separating mammalian orders (12, 13). This analysissuggests that émergence of the P/3 domain at the outset of verte-brate évolution may be associated with the appearance of a newmechanism of régulation of PARL. We hâve recently shown thatthis part of the PARL molécule undergoes two consécutive cleav-age events, termed a and /3. The proximal a-cleavage is a consti-tutive processing associated with the protein import in the mito-chondria, whereas the distal /3-cleavage is regulated through amechanism of proteolysis requiringPARLactivity supplied in trans(11). Whether this cleavage occurs in vivo is unknown. In addition,its mechanism of régulation and its functional significance remainunexplored.

Results

Human PARL Is Subjected to /3-Cleavage in Vivo. PARL transfectedin HEK 293 cells is cleaved at position Ser77-Ala7X, which mapswithin the vertebrate-specific P/3 domain (11), suggesting that, invivo, the rhomboid protease may undergo the same processing. Toaddress this possibility, we generated a polyclonal antibody againsta peptide spanning the C terminus of PARL (anti-PARL-C-Term;Fig. \A). This spécifie antibody (Fig. lfi) was used to immunopre-cipitate endogenous PARL from lysates of mitochondria isolatedfrom human placenta. Using antibodies recognizing the N-terminaland C-terminal régions of PARL (Fig. 1/1), we then examined thecleavage of the endogenous protein relative to the transfectedPARL-FCT by epitope mapping. Data showed two bands whosemobility is, as expected, slightly higher than that of the FLAG-tagged positive control (Fig. \C Upper). Although both forms areimmunoreactive against anti-PARL-C-Term, only the slower mi-grating band was positive to anti-PARL-N-Term, indicating thatthe corresponding epitope was absent in the faster migrating band(Fig. IC). Thèse data strongly suggest that endogenous PARL Nterminus undergoes /3-cleavage, indicating that this processing maymechanistically coordinate the function of the rhomboid protease

Author contributions: D.V.J. and L.X. contributed equally to this work; H.M.M. and L.P.designed research; D.V.J., L.X., M.-C.L., S.B., R.Z., E.A.B., H.M.M., and L.P. performedresearch; H.M.M. and L.P. contributed new reagents/analytictools; E.A.B., H.M.M., and L.P.analyzed data; and H.M.M. and L.P. wrote the paper.

Conflict of interest statement: E.A.B. is an employée and stockholder of Century 21stBiochemicals. This Company sells mass spectrometry analysis and antibody productionservices.

This article isa PNAS direct submission. W.N. isaguesteditor invited by the Editorial Board.

Abbreviations: LC/MS, liquid chromatography/mass spectrometry; MAMP, mature mito-chondrial PARL; PAO", PARL C-terminal product (of ^-cleavage); PARL, presenilin-associ-ated rhomboid-like (protein); PARL-FCT, PARL-FLAG-CTe,mmui.5To whom correspondence may be addressed. E-mail; hmcbride@ottawaheart.ca orluca.pellegrini@crulrg.ulaval.ca.

© 2006 by The National Academy of Sciences of the USA

18562-18567 | PNAS | December 5,2006 | vol. 103 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.0604983103

J PARL 42 kDa

1 MAMP 36 kDa

L

I PARL-N-Term epitope

IPanti-PARL-C-Term

] PACT 33kDa

PARL-C-Term epitope

Peptide PARL-C-Term : —

Lysate fromPARL-FCT

transfectedcells

Lysate fromhuman placenta

mitochondria

TransfectedPARL-FLAG-CT • I

I endogenousMAMP-FLAG-CT • • • < ] MAMP• mm

Transfected

MAMP-FLAG-CT *•35 kDa -

PACT-FLAG-CT •

TransfectedPARL-FLAG-CT

MAMP-FLAG-CT »•35 kDa —

WB anti-PARL-N-term

IP anti-PARL-C-Term

PARL-FCT Humantransfected placenta

cells mitochondria

f l endogenous< ] MAMP

< 3 PACT

WB anti-PARL-C-Term

WB anti-PARL-N-Term

endogenous] MAMP

Fig. 1. Cleavageof PARL P/3 domain in vivo. (A) Schematic représentation ofthe a- and (3-cleaved forms of PARL, MAMP, and the PARL C-terminal productof |3-cleavage (PACT). The locations of the epitopes recognized by the anti-PARL.-N-Term and anti-PARL-C-Term antibodies are indicated. Small blacksquares depict the seven transmembrane helixes of PARL. (S) Specificity of theanti-PARL-C-Term antibody. The anti-PARL-C-Term antibody specifically im-munoprecipitates (IP) PARL-FLAG-Cjerminus (PARL-FCT) from HEK 293-transfected cells as well as endogenous PARL from mitochondrial lysates ofhuman placenta. The specificity was addressed by preadsorbing the antiserawith the synthetic peptide PARL-C-Term used to generate the correspondingantibody. WB, Western blotting. ( 0 Endogenous PARL is subjected to N-terminal )3-cleavage as observed for transfected PARL. Transfected PARL-FCTand endogenous PARL were immunoprecipitated with anti-PARL-C-Term andsubjected to epitope mapping. (L/pper) Both the transfected and endogenousPARL. are présent in two forms, MAMP and PACT, which migrate according tothe predicted molecular mass shown in A. The lower band labeled as PACT-FLAG-CT corresponds to the product of p-cleavage at residue Ser77-Arg78 (11).Note the slight différence in mobility between transfected and endogenousPACT, which is caused by the présence/absence of the FLAG tag. (Lower)Transfected and endogenous PACT lack the N-Term epitope because of N-terminal 0-cleavage. Asterisks indicate nonspecific cleavages.

Endogenous and Transfected PARL Are Hyperphosphorylated at theVertebrate-Specific P/3 Domain. To investigate the mechanism ofrégulation of PARL /3-cleavage in vivo, we conducted mass spec-trometric studies to identify posttranslational modifications on theP/3 domain (14, 15). PARL was immunoprecipitated from lysates

obtained from 250 mg of solubilized human placenta mitochondria,it was digested, and the peptides were subjected to LC/MS analysis.Data showed that >35% of the entire protein was covered (Table1, which is published as supporting information on the PNAS website), with two molecular ions spanning nearly the entire P/3 domainof the mitochondrial mature form of PARL, MAMP (Fig. IÂ). Ionm/z 1072.932+ corresponded to a triple-phosphorylated 6()VE-PRRSDPGTSGEAYKR76 peptide, which maps between the a-and j3-cleavage sites; ion m/z 1138.132+ corresponded instead to anunmodified peptide spanning the /3-cleavage site and its distalrégion (77SALIPPVEETVFYPSPYPIR%; Fig. 2 A and E).

To investigate whether transfected PARL is also phosphorylated,we overexpressed PARL-FCT in HEK 293 cells. The protein wasimmunoprecipitated with anti-FLAG to isolate the transfectedprotein. It was unlikely that endogenous PARL was copurifiedduring this step because coimmunoprecipitation studies withPARL constructs harboring différent tags did not reveal ho-modimers or oligomers (data not shown). The a-cleaved form ofPARL, MAMP (Fig. \A), was then isolated by gel electrophoresis,digested, and subjected to LC/MS analysis. More than 51% of theprotein was covered (Table 2, which is published as supportinginformation on the PNAS web site). Within this peptide data set wealso observed a triple-phosphorylated 56APRKVEPRRSDPGTS-GEAYKR76 peptide, molecular ion m/z 866.393+ (Fig. 25), whichoverlaps with most of the triple- phosphorylated m/z 1072.932+

identified during the analysis of the endogenous PARL. Molecularion m/z 1138.132+, 77SALIPPVEETVFYPSPYPIR%, was alsofound (Table 2), indicating that sample préparation and analyseswere performed under comparable expérimental conditions. Sim-ilar results were also obtained from PARL-FCT isolated fromtransfected HeLa cells (data not shown).

To refine thèse results, we subjected ion m/z 866.393+ to tandemMS analysis. Data showed a séries of three water and phosphoricacid losses as the primary detected fragments (Fig. 2 D and E andTable 3, which is published as supporting information on the PNASweb site), consistent with the fragmentation pattern of a peptidewith phosphorylated Ser and Thr, rather than Tyr residues. Addi-tionally, the nonphosphorylated y3 ion and the Y immonium ionbut not their corresponding phosphorylated species were detected,indicating lack of phosphorylation at Tyr-74 of PARL. Further-more, the N-terminal ion bi2-H3PO4, an internai ion séries(GTSG-2H3PO4, PGTS-2H3PO4, DPGTSG-2H3PO4), and theC-terminal ion yis-HjPCU indicate phosphorylation at Ser-65,Thr-69, and Ser-70 (Fig. 2 D and E and Table 3). This conclusionwas further supported by the lack of phosphorylated peptides in thedata set obtained from LC/MS analysis of a transfected PARLmutant bearing Ala substitutions at thèse residues (Fig. 2 C and Eand data not shown). A monophosphorylated peptide spanningSer-65, Thr-69, and Ser-70 was detected in vivo as well as in vitro(Table 1); however, its relative abundance was very low comparedwith the triple- phosphorylated form (data not shown), indicatingthat most of the a-cleaved form of PARL is phosphorylated. Weconclude that the vertebrate-specific P/3 domain of endogenous andtransfected PARL is phosphorylated at residues Ser-65, Thr-69,and Ser-70, and, by implication, that this modification has the samefunction in vivo and in vitro.

The Phosphorylated Pji Domain Is Exposed to the Matrix. To déter-mine the localization of the phosphorylated, vertebrate-specificP/3 domain of PARL, we investigated the topology of the protein.To this end, we used a PARL construct with a hemagglutinin(HA) tag inserted at its N terminus, at position 91, and a FLAGtag at its C terminus (PARL-HA-FCT; Fig. 3C). We perme-abilized PARL-H A-FCT-transfected HeLa cells with increasingamounts of digitonin, and we performed immunofluorescencewith antibodies against either the FLAG or HA tag. At lowconcentrations of digitonin, only the plasma membrane waspermeabilized, allowing the outer membrane receptor Tom20 to

Jeyaraju et a/. PNAS I December 5,2006 I vol. 103 I no. 49 18563

B'

1072.93

mf\ i = j i i a, . • l i i l i , l ! . . i i l . H . . 1 , 1 . . . , X.040 8B0 920 1000 1040 1080 1120 1160 m 500 700 900 1100 1300mA 600 800 m»

EnûogenousMAMP

(A)

TransfectedMAMP

FLAG-CT(B)

TransfecledMAMP

FLAG-CTmutant STS/AAA

(C)

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53GFRKAPRKVEPRRSDPGTSGEAYKRSALIPPVEETVFYPSPYPIRSLIK1CO P|i-domain

77SALIPPVEETVFYPSPYPIR (unmodified)

(+ 3 phosphates)

ion m/z 1138.13 •"

ion m/z 1072.93 2* 60VEPRRSDPGTSGEAYKR

ion m/z 866 39 3* 56APRKVEPRRSpPGTSGEAYKR

ion m/z 686.71 2* 56APRKVEPRRSDP

ion m/z 307 20 * 67PGTS

ion m/z 603 30 esADPGAAGEAYK

(+ 3 phosphates)

(- 1 phosphate, - 1 water) I Tandem MSI analysé of

(- 2 phosphate. - 2 water) | ion m/z 866.39 3*(D)

(unmodified)

Fig. 2. Phosphorylation of PARL P/3 domain at Ser-65, Thr-69, and Ser-70 in vivo and in vitro. {A) Phosphorylation of endogenous PARL. LC/MS analysis of PARLfrom mitochondria purified from human placenta is shown. The protein was immunoprecipitated with the anti-PARL-C-Term antibody (Fig. 1), digested, andsubjected to LC/MS analysis. (Left) Molecular ion m/z 1072.932+, which corresponds to a triple-phosphorylated 60VEPRRSDPGTSGEAYKR76 peptide mappingbetween PARL a- and /3-cleavage sites (Fig. 1 A) (11). (flighf) Ion m/z 1138.132\ corresponding to the unmodified "SAUPPVEETVFYPSPYPIR96 peptide, which alsomaps on the vertebrate-specif ic P(3 domain of PARL. More than 35% of the mature form of PARL (MAMP; Fig. 1 A) could be found through this analysis; thecomplète list of the ions is shown in Table 1. The identity of each peptide was determined manually and with a Bayesian reconstruction algorithm as well assearching against both theoretical peptide and fragmentation data from the PARL séquence. (8) Phosphorylation of transfected PARL-FCT. LC/MS analysis oftransfected PARL-FCT purified from HEK293 cells is shown. MAMP-FLAG-Grerminus (MAMP-FLAG-CT) was immunoprecipitated with anti-FLAG monoclonalantibody, purified by gel electrophoresis, digested, and analyzed by LC/MS analysis. The triple-phosphorylated 56APRKVEPRRSDPGTSGEAYKR76 peptide, ion m/z866.393+, is indicated. More than 51 % of MAMP séquence could be found through this analysis; the complète list of ions is shown in Table 2. (C) PARL mutantS65A/T69A/S70A is not phosphorylated. LC/MS analysis of transfected PARL-FCT mutant AAA purified from HEK 293 cells is presented. The data show ion mlz603.32+, corresponding to the unphosphorylated 6SADPGAAGEAYK75 peptide. Note that no phosphorylated peptides encompassing the Pf3 domain of thismutant protein were found (data not shown). (D) Tandem MS analysis of phosphorylated PARL. Ion m/z 866.393+ was fragmented to detect peptides that,through the loss of phosphate group(s) and/or water (-H3PO4), finely map phosphorylation at Ser-65, Thr-69, and Ser-70. The N-terminal ion m/z 686,7124

(56APRKVEPRR5DP-H3PO4) and m/z 307.2' (67PG7S-2H3PO4) are shown in (£). The complète list of molecular ions is shown in Table 3. The identity andphosphorylation state of each peptide were determined by both manual interprétation of the spectra and a Mascot search of ail of the enhanced product ionscans. (F) Schematic représentation summarizing the results showing phosphorylation of endogenous and transfected PARL at residue Ser-65, Thr-69, and Ser-70.

be efficiently recognized by its antibody in =100% of cells (Fig.3 A and B). As the digitonin concentration increased, first, 70%of cells were efficiently labeled with the FLAG along with theintermembrane space marker cytochrome c. In contrast, the HAepitope was efficiently labeled (=60% of cells) only on ircatmentwith the highest concentrations of digitonin, which appearedconeomitantly with the matrix marker peroxidoreductase3/sp-22 (16). Thèse data indicate that the C-terminal domain ofPARL is located in the intermembrane space, whereas theN-terminal phosphorylated P/3 domain is exposed in the mito-chondrial matrix, thereby correcting our previous examination ofPARL lopology using immunogold-labeled EM sections (11).

Phosphorylation of the P/3 Domain Régulâtes /3-Cleavage. Givcn theproximily of the phosphorylated rcsidues to the j8-cleavage site, weinvestigaled whether this modification could hâve a regulatoryfunction on the cleavage itself. We therefore mutated residuesSer-65, Thr-69, and Ser-70 to alanine, to abolish phosphorylation,and to aspartic acid, which is commonly used to mimic phosphor-ylation by introducing a négative charge (17,18). We then tested theeffect of thèse mutations on /3-cleavage, and we found that Aspsubstitutions of ail three phosphorylated amino acids led to stronglyreduced levels of /3-cleavage (Fig. 44). Impaired (3-cleavage wasalso observed with two double Asp mutants, S65D/T69A/S70D andS65A/T69D/S70D (Fig. 44) but not with single substitutions (data

not shown), suggesting an additive inhibitory effect of each phos-phorylated amino acid on /3-cleavage. To demonstrate further theinhibitory effect of Asp substitutions on (3-cleavage, we performeda large-scale mutagenesis screening to identify PARL mutants withconstitutive /3-cleavage. A mutant carrying the K4EETV87 deletionin the P/3 domain, A84-87, showed dramatically increased /3-cleav-age (Fig. 4 B and C). However, in this mutant, Asp substitutions inail three phosphorylated residues dominantly reestablished block ofj8-cleavage (Fig. AD). Notably, none of the PARL mutants analyzedin this work with deletions and/or substitutions on the P/3 domainhad compromised protease activity, as indicated by their ability tocleave in trans acatalytically dead (S277G) PARLprotease (Fig. \Eand data not shown). Therefore, lack of /3-cleavage of the mutantmimicking constitutive phosphorylation of PARL, DDD, is nol theresuit of loss of proteolytic activity. This observation also impliesthat the import and insertion were not compromised, as furtherconfirmed in digitonin-permeabilization experiments (data notshown). We conclude that the stable phosphorylation of residuesSer-65, Thr-69, and Ser-70 inhibits PARL /3-cleavage.

/J-Cleavage Médiates PARL Activity in Mitochondrial Morphology. Wenext examined the rôle of /3-cleavage and phosphorylation onmitochondrial morphology. Transient expression of wild-typePARL-FCT in HeLa cells resulted in a dramatic increase inmitochondrial fragmentation (Fig. 5 A and B). Similar resulls were

18564 | www.pnas.org/cgi/doi/10.1073/pnas.0604983103 Jeyaraju ef al.

0.012

1

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IP anti-FLAG, WB anti-FLAG IP anti-FLAG, WB anti-FLAG

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Fig. i. Localization of PARL P0 domain in the matrix. {A) HeLa cells weretransfected with a construct expressing PARL-HA-FCT (see scheme in O. fixed,and permeabilized with the indicated concentrations of digitonin. For eachcondition, cells were coimmunostained with anti-FLAG or anti-HA, and anti-Tora'O (to label the outer membrane), or anti-cytochrome c (to label theintermembrane space), or anti-peroxiredoxin 3 (to label the matrix). (8)Quantitation of the experiments shown in A (O Scheme summarizing thetopology of PARL.

observed with the S65A/T69A/S70A mutant and the constitutivelyj3-cleaved PARL protein, A84-87. By contrast, similar levels ofexpiession of the S65D/T69D/S70D mutant (Fig. 5C and data notshown) did not resuit in significant mitochondrial fragmentation(Fig. 5), suggesting that transient expression of the nonphospho-rylated, j3-cleaved form of PARL leads to fragmented mitochon-dria. To investigate this observation further, we transfected mutantsin which j3-cleavage was abolished by removing (PARLA75-79) ormutating (PARLL79u) the cleavage site. Expression of either pro-tein did not induce mitochondrial fragmentation (Fig. 5 A and B),further indicating that /3-cleavage is required to initiate this process.

Cleavage of PARL at the /3-site libérâtes the Pj3 peptide, a 25-aapeplide that can target the nucleus when released to the cytosol(11). To investigate whether the liberated Pj3 peptide is functionallyimplicated in the initiation of mitochondrial fragmentation, wetransfected mutants in which we deleted parts of its séquence,A56-59 and A58-61 (Fig. 4fl), and we analyzed the morphology ofthe mitochondria. Data showed that neither deletion impaired/3-cleavage and mitochondrial fragmentation (Fig. 5), indicatingthat the function of the P/3 peptide is independent of the initiationof PARL-induced mitochondrial fragmentation.

DiscussionConsidérable mechanistic and functional information on the mi-tochondrial rhomboid protease PCP1P in yeast is available (7,8,19,

PARLMyc-CT

- Wild type

- S277G

- STS 65-69-70 AAA

- STS 65-69-70 DOD

- A 84-87 (EE TV]

- A 87-90 (VFYP)

+ PARL-FLAG-CT S277G

IP anti-FLAG, WBanti-FLAG

Fig. 4. Substitutions mimicking phosphorylation at Ser-65, Thr-69, andSer-70 inhibit PARL 0-cleavage. (A) Constructs expressing the indicated mu-tant PARL protein were transfected in either HEK 293 or HeLa cells. The effectof mutations abolishing (Ala) or mimicking (Asp) phosphorylation (17, 18) ismonitored bytheamountof /3-cleaved form of PARL detected, PACT (Fig. 1/4).Note that Asp but not Ala substitutions block /3-cleavage. IP, immunoprecipi-tation; WB, Western blotting. (B) Scheme of the deletions (A) within PARL P/3domain that hâve been tested in this work. (O The A84-87 mutant is consti-tutively cleaved at the (3-cleavage site. (D) Asp substitutions at positionsSer-65, Thr-69, and Ser-70 in the A84-87 dominantly reestablish the block ofp-cleavage. (£) Ala and Asp substitutions at the phosphorylated Ser-65, Thr-69,and Ser-70 residues do not affect PARL protease activity. HEK 293 cells werecotransfected with a catalytically dead PARL protein (PARL-Myc-CT S277G)and the indicated FLAG-tagged mutant, whose enzymatic activity is moni-tored by itsability tocleavethe inactive PARLintransand produce PACT (11).

20). On the other hand, the rôle of its mammalian ortholog PARLappears less clear. In higher organisms, dynamic changes in mito-chondrial shape hâve been implicated in programmed cell death(4-6), a process that emerged late during metazoan évolution.Therefore, the machinery of mitochondrial fusion and fission islikely to be regulated by mechanisms additional to those for yeast.Our results implicate phosphorylation and cleavage of the P/Bdomain of PARL in mitochondrial morphology. Because thisdomain is vertebrate-specific (11), this processing apparently is aregulatory mechanism that emerged during vertebrate évolution.

Jeyaraju et al. PNAS | December 5,2006 | vol. 103 | no. 49 | 18565

B "

I ipOCT VFP rypc

cAAA DDD

M M

HeLa tell

DO

L79E

• •

IP

L ? 9 E 7S-79

PARL FCT

\ A A A75-79 81-87 S6-S9SB-61

nti-fLAG. WB anti-FLAG

84-87 S6 S9 bfl-6

•< MAMP< PACT

Fig. 5. Cleavage of the vertebrate-specific P/} domain is required to médiatePARL activity in mitochondrial morphology. (A) HeLa cells were transfectedwith the indicated PARL-FCT constructs, fixed, permeabilized, and stainedwith monoclonal anti-FLAG (green) and polydonal anti-Tom20 (red). Imageswere taken with the Olympus FV1000confocal microscope. (Scalebars, 5/im.)(B) The mitochondrial morphologies of the wild-type and PARL mutantsshown in A were quantified from three independent experiments, counting100 cells per experiment. (O Cells in A express similar levels of transfectedproteins. Anti-FLAG immunoprecipitation (IP) and Western blot (WB) analysisof the FLAG-tagged PARL constructs used in A are shown. Equal amounts ofimmunoprecipitated protein were loaded. Note that in separate experiments,we verified that the immunoprecipitation efficiently depleted ail of thetran;,fected protein, validating this comparison (data not shown).

Recently, it has been shown that deletion of Pari in the mouseresulted in prématuré postnatal death (21), which correlatcd withreduced levels of cleaved OPA1. OPÀ1 has been shown to beinvolved in the régulation of the so-called "cristae rcmodeling"pathway of apoptosis (21, 22) and the régulation of mitochondrialfusion (23). Although PARL was shown to cleave OPA1, onlyminor changes in mitochondrial morphology were observed inPari'1' fibroblasts (21), which indicates that PARL is not directlyrequired for mitochondrial fusion (21). Therefore, the expression ofa cleaved form of PARL appears to be a gain of function, leadingto the fragmented morphology we hâve observed in this work.Because PARL-induced fragmentation dépends on its phosphor-ylation and cleavage, the functional outcome of PARL expressionon steady-state mitochondrial morphology is likely to be regulatedby the abundance and activity of the yet-unidentified PARL kinasephosphatase, and protease. Thus, apparently disaccording results

may arise from a complex set of regulators thaï could be expressedat différent levels in différent tissues and cells.

To date, the only réversible phosphorylation/dephosphorylationevents known to occur within the mitochondrial inlermembranespace or matrix compartment are limited to the El subunits of thepyruvate and branched-chain a-ketoacid dehydrogenase complexes(24, 25). The identification of the kinase/phosphatase couple thatrégulâtes PARL cleavage will be of critical importance to under-stand the régulation of mitochondrial morphology in différenttissues. Moreover, the discovery of a rôle for phosphorylation inmitochondrial dynamics could also provide an explanation for thecontrasting reports on the effect of proteins controlling mitochon-drial dynamics. For example, expression of OPA1 increased mito-chondrial fusion in mouse embryonic fibroblasts and in NIH 3T3fibroblasts, whereas it resulted in dramatic fragmentation in COS-7cells (23, 26, 27). Similarly, expression of DRP1 did not lead tofission in most cell types, but it was reported to fragment organellesin an inducible HeLa cell Une (28). Finally, it is tempting tospeculate that tissue selectivity of the clinical phenotype of domi-nant optic atrophy and Charcot-Marie-Tooth Ha, caused by mu-tations in OPA1 and MFN2 respectively, could similarly be aconséquence of differential phosphorylation (6).

/3-Cleavage libérales a 25-aa nuclear-targeted peptide termed PjSpeptide (11). Topology of PARL now shows that this peptide isgenerated in the matrix. A récent MS study has demonstrated theexistence of a constant eff lux of a large number of peptides fromthe mitochondria (29). Thèse peptides, which originate from thecleavage of proteins localized mainly in the malrix and innermembrane, range in size from 6 to 27 aa, and they are extruded tothe cytosol in an ATP- and temperature-dependent manner (29).Whether the P/3 peptide can be exported from the matrix to thecytosol remains to be demonstrated. However, the existence ofspecialized machinery for the export of matrix peptides of similarsize supports this possibility. The fact that an intégral Pj3 séquenceis not required for PARL-mediated mitochondrial fragmentation(Fig. 5A) is consistent with the possibility that the P/3 peptidemédiates milochondria-to-nucleus signaling (11).

Materials and MethodsCell Lines and Antibodies. HEK 293 and HeLa cells were purchasedfrom American Type Culture Collection (Manassas, VA) andmaintained under standard cell culture conditions. Cells weretransfected at 40% confluence with FuGENE 6. The antibodiesused in this work were: polyclonal anti-GFP (Clontech, MountainView, CA), monoclonal anti-GFP (Invitrogen, Carlsbad, CA),monoclonal anti-cytochromec and polyclonal anti-DsRed (PharM-ingen, San Diego, CA), monoclonal anti-HA (Covance, Denver,PA), mouse M2 monoclonal and rabbit polyclonal anti-FLAG.Polyclonal antibodies against the matrix marker peroxiredoxin 3(30) were raised in rabbits against the recombinant human GST-PRDX3 protein following standard immunization protocols. Sé-rum was tested for specificity by preadsorption wilh the antigen.Anti-PARL-N-Term antibody has been already described in réf. 11.Anti-PARL-C-Term was raised against a peptide spanning the last12 aa of PARL conjugated to keyhole limpet hemocyanin, accord-ing to standard immunization protocols. Immunoprecipitationsdone with this antiserum were performed by covalently couplingthis antiserum to protein A-agarose beads.

Constructs. The vector used to express the human PARL proteinin mammalian cells was pcDNA3. The PARL-FCT and PARL-HA-FCT constructs hâve been described in réf. 11. Mutants ofthèse PARL constructs were obtained by site-directed mutagen-esis. Ail mutations were confirmed by DNA sequencing.

PARL Cleavage Analysis. Cells were transfected with the indicatedPARL construct, grown for 24-36 h (unless otherwise indi-cated), washed with Dulbecco's PBS, and lysed in RIPA buffer

18566 | www.pnas.org/cgi/doi/l0.1073/pnas.0604983103 Jeyaraju étal.

containing a mixture of protease inhibitors and 1 mM sodiumorthovanadate. Immunoprecipitations were performed withanti-FLAG-M2 monoclonal antibody or anti-PARL-C-Term at4°C overnight as described in réf. 31. Immunocomplexes werewaslied four times with RIPA and denatured at 85°C for 4 minin Laemmli buffer. Samples were fractionated by SDS/PAGE ona 4-12% (wt/vol) 2-[bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)l,3-propanediol gel, blotted on PVDF membranes, im-munodetected by Western blot analysis, and imaged by using theVersadoc System (Bio-Rad, Hercules, CA). For MS analysis, gelswere stained with colloidal Coomassie blue. Stained bands wereexcised and stored at -70°C until ready for LC/MS analysis.

MS. Samples were prepared as described in réf. 15. For capillaryLC/MS, samples were analyzed by directed infusion of chromato-graphically separated components on an MDS Sciex QStar XLmass spectrometer (Applied Biosystems, Foster City, CA) inter-faced with an Ultimate micropump (LC Packings, Sunnyvale, CA).Capillary columns (150 jun X 100 mm) were packed in-house withMajic C-18 reversed-phase packing material. A 150-min continuousgradient was used for séparation with buffer A [2% ACN (vol/vol)/0.1% formic acid/0.01% TFA] followed by buffer B [10% isopropylalcohol (vol/vol)/80% ACN (vol/vol)/0.1% formic acid/0.01%TFA]. Dried samples were resuspended in A buffer and loadeddirectly on the capillary column. Samples were sprayed at 4500Vand MS along with tandem MS data were acquired on the f ly byusing the Analyst QS software (Applied Biosystems).

Data Analysis. Résultant data were reconstructed manually and witha Bayesian reconstruction algorithm, and they were searchedagainst both theoretical peptide and fragmentation data from thePARL séquence. Tandem data were used for web-based scarcheswith Mascot (Matrix Science Ltd., London, U.K.). Matching tan-dem data were verified manually.

Mitochondrial Morphology Analysis. Fluorescence imaging. HeLa cellswere transfected as described in réf. 32. To stain mitochondria withpotentially sensitive dyes, cells were incubated with 50 nM Mit-oFluor Red 589 (Invitrogen) at 37°C for 15 min before imaging. Forquantification of mitochondrial phenotypes, images were obtainedwith an Olympus 1X70 microscope (Olympus Canada, Markham,ON, Canada) through a 100X objective U Plan Apochromat, NA1.35-0.50 objective, excited at 500 nm (yellow fluorescent protein;YFP), 434 nm (cyan fluorescent protein; CFP), and 589 nm(MitoFluor Red 589) with the Polychrome IV monochrometer.The emitted light was filtered through a triple CFP/YFP/DsRed

pass filter. Acquired images and multichannel overlaying were doncwith TillVision IV software (TILL Photonics, Pleasanton, CA).Quantification of mitochondrial morphology was done according tomitochondrial length to width ratio. When the ratio was >3:1, themitochondria were classified as tubular and rod-shaped, and whenthe ratio was <3:1, mitochondria were classified as fragmented.Data were obtained from 100 cells from each condition, andstandard déviations were calculated from three independent ex-periments. High-resolution images were obtained from samplestransfected with PARL-FCT constructs and costained with mono-clonal anti-FLAG and polyclonal anti-Tom20 by using an OlympusFV1000 confocal microscope (Olympus Canada). A 100X U PlanApochromat objective NA 1.45 was used along with the argon laserto excite the 488 nm secondary Alexa 488 and the Red HeNe laserfor Alexa 647 secondary antibody. The images shown are from10-20 compressed Z stacks taken in 0.12-^im intervais to capture«=2-;um sections of the cell by using Kalman averaging of two scanseach (33).Digitonin permeabilization. Digitonin was recrystallized as describedin réf. 34 and resuspended in PBS. Transfected cells were fixed in4% paraformaldehyde and permeabilized with increasing concen-trations of digitonin before standard immunofluorescence wasperformed with the indicated antibody as described previously. Thedata were quantified from 100 cells in three independent experi-ments (35).

Isolation of Mitochondria. Fresh human placenta was obtained withappropriate permission, eut into fragments, and washed with PBSbefore decanting into 2 volumes per volume of mitochondrialisolation buffer (220 mM mannitol/68 mM sucrose/20 mM Hepes,pH 7.4/80 mM KCI/0.5 mM EGTA/2 mM MgOAc/protease inhib-itors). The tissue was homogenized by using a Waring blender (ColePalmer, Ansou, QC, Canada), and mitochondria were isolated bystandard differential centrifugation. The mitochondrial pellet wasresuspended in 100 ml of mitochondrial isolation buffer with 10%glycerol, then it was snap frozen and stored at -80°C.

We thank Dr. Gordon Shore (McGill University, Montréal, QC, Canada)for the anti-Tom20 antibody and Dr. Jordan Fishman (21st CenturyBiochemicals) for assistance in the génération of antibodies. This work wassupported by grants from the Natural Sciences and Engineering ResearchCouncil of Canada, the Canada Foundation for Innovation, and the Centrede Recherche Université Laval Robert-Giffard (to L.P.); grants from theCanadian Institutes of Health Research and the Canada Foundation forInnovation (to the H.M.M. laboratory); a Fonds de la Recherche en Santédu Québec Junior-2 Scholarship (to L.P.); and a Canadian Institutes ofHealth Research New Investigator Award (to H.M.M.).

1. Meeuscn SL. Nunnari .1 (2005) Curr Opin Cell Biol 17:389-394. 19.2. Okamoto K, Shaw JM (2005) Annu Rev Genêt 3<M03-536. 20.3. Yaffe Ml' (2003) Nal Cell Biol 5:497-4'».4. Bossy-Wetzel li, Harsoum Ml, Godzik A, Schwarzcnbachcr R, Liplon SA (2003) Curr Opin 21.

Cell Biol 15:706-716.5. Karbowski M, Youlc R.I (2003) Cell Dailh Differ 10:870-880. 22.6. Chen H, Chan DC (2005) Ilum Mnl Genêt 14(Suppl 2):R283-R289.7. I lerlan M, Vogcl F, Bomhovd C, Neupcrt W, Reichert AS (2003)7BmIChem 278:277X1-27788. 23.8. McQuihban (iA, Saurya S, Frecman M (2003) Nature 423:537-541.9. l'ellcgrini L, Passer li.1. Candies M, Leflerov I, (ïanjei JK, Fowlkes 1ÎJ, Koonin LiV, 24.

D'Adamio L (2001) J Alzheimers Dis 3:181-190.10. Koonin liV, Makarova KS, Rogozin I», Davidovic L, Utellier MC, Pcllcgrini L (2003) 25.

Centime Biol 4:1-12.11. Sik A, Passer RI, Koonin lïV, Pcllcgrini L (2004) J Biol Chem 279:15323-15329. 26.12. Waterslon RH, Lindblad-ïoh K, lîirncy li, Rogera .1, Abril JF. Agarwal P, Agarwala R, 27.

Ainscough R, Alexanclcrsson M, An P, et al. (2002) Nature 420:520-562.13. OgurtSOV AY, Sunyacv S, Kondrashov AS (2004) Génome Iles 14:1610-1616. 28.14. Borchers Cil, Thapar R, Pctrolchenko liV, Tories MP, Spcir JP, Easterling M, Domlnski

Zi, Marzlulï WF (2006) Vroc Natl Acad Sci USA 103:3094-3099. 29.15. Perlman DU, Herg LiA, O'Connor PH, Coslello CE, Hu J (2005) Proc Natl Acad Sci USA

102:9020-9025. 30.16. Walabc S, Kohno II, Kouyama II, Hiroi T, Yago N, NakazawaT (1994)./ Biochem (Tokyo) 31.

115:648-654. 32.17. Iyer D, Chang I), Marx .1, Wei L, Oison IiN, Parmacek MS, Balasubramanyam A, Schwarlz 33.

R.I (2006) l'mc Natl Acad Sci USA 103:4516-4521.18. l e e SY, Wcnk MR, Kim Y, Nairn AC, De Camilli P (2004) Proc Natl Acad Sci USA 34.

101:546-551. 35.

Merlan M, Hornhovd C, Hell K, Neuperl W, Rcichert AS (2004)./ Cell Biol 165:167-173.Sesaki H, Southard SM, Ilobbs AH, Jensen Rli (2003) Biochem Biophys lies Commun308:276-283.Cipolat S, Rudka '1', Hartmann I), Cosla V, Serneels L, Craessaerts K, Mclzger K, FrezzaC, Annaert W, D'Adamio L, et al. (2006) Cell 126:163-175.Frezza C, Cipolal S, Marlins de Brito O, Micaroni M, Beznoussenko (ÎV, Rudka T, BarloliD, Polishuck RS, Danial NN, De Slrooper H, Scorrano L (2006) Cell 126:177-189.Cipolat S, Marlins de Brito O, Dal Zilio », Scorrano L (2004) Proc Natl Acad Sci USA101:15927-15932.I larris R A, I lawes ,1W, Popov KM, Zhao Y, Shimomura Y, Salo J, Jaskiewicz .1, Ilurlcy 11)(1997) Adv Enzyme Kegul 37:271-293.Roche TE, Baker JC, Yan X, Hiromasa Y, (iong X, l'eng T, Dong J, Turkan A, Kaslen SA(2001) ProK Nucleic Acid Res Mol Biol 70:33-75.Misaka T, Miyashita T, Kubo Y (2002) J Biol Chem 277:15834-15842.Oliehon A, Lmorine LJ, Descoins li, Pelloquin L, Brichese L, Cïas N, (iuillou li, DclcurcC, Valette A, Ilamel CP, et al. (2002) FEBS I.ett 523:171-176.Szabadkai (i, Simoni AM, Chami M, Wieckowski MR, Youlc R.I, Rizzuto R (2004) Mol Cell16:59-68.Augustin S, Nolden M, Muller S, Hardi O, Arnold I, Langer T (2005) ./ Biol Chem280:2691-2699.Wonsey DR, Zeller Kl, Dang CV (2002) Proc Natl Acad Sci USA 99:6649-6654.Pellegrini L, Pa«er B.I, Tabalon M, Ganjci JK, D'Adamio L (1999) J Biol Chan 274:21011-21016.Harder Z, Zunino R, McBridc H (2004) Curr Biol 14:340-345.Neuspiel M, Zunino R, Gangaraju S, Rippslein P, McBridc HM (2005)7 Biol Chem280:25060-25070.Kun li, Kirsten li, Piper WN (1979) Mclhods Enzymol 55:115-118.Otera H, Ohsakaya S, Nagaura Z, Ishihara N, Mihara K (2005) EMBO J 24:1375-1386,

Jeyaraju et al. PNAS | December 5,2006 | vol. 103 no. 49 | 18567

Perspectives

Identification of the molecular machinery regulating PARL P-cleavage

PARL kinase and phosphatase are the gatekeeper of PARL (3-cleavage. Thus their

characterization will lead to to the discovery of the pathway implicated in the régulation of

mitochondria remodelling via PARL. The identification of PARL kinase and phosphatase is ail

the more important in considération of the limited knowledge we hâve on the rôle of réversible

phosphorylation in the mitochondrion. To date, the only known example of this process within

the mitochondnal intermembrane space or matrix compartment are limited to the El subunits of

the pyruvate and branched-chain a-ketoacid dehydrogenase complexes (Harris et al 1997, Roche

et al 2001). Only a handful of kinases hâve been shown to réside inside the organelle (Horbinski

& Chu 2005). They are the branched-chain a-ketoacid dehydrogenase kinase, pyruvate

dehydrogenase kinase, PKA, few isoforms of protein kinase C (PKC), GSK 3p\ CK2, and

PINK1. Such small number of mitochondnal kinases (and phosphatases), most of which do not

hâve known substrates (Horbinski & Chu 2005, Salvi et al 2005), indicate how little we know

about the rôle of thèse enzymes in the régulation of fonctions occurring in the inner membrane

and in the matrix, such as ATP synthesis, mtDNA replication, ROS production and scavenging,

cristae remodeling and cytochrome c release during apoptosis. Understanding how thèse

processes are regulated by protein phosphorylation has become ail the more compelling since the

récent discovery that mutations in mitochondrial kinases and phosphatases are linked to major

human diseases, such as Parkinson's (Valente et al 2004) and diabètes (Pagliarini et al 2005).

Identification of PARLase

As for PARL kinase and phosphatase,, the identification of the protease that actually exécutes P-

cleavage, PARLase, is critically important. PARLase is indeed likely to be a substrate of PARL

(Sik et al 2004). In addition to completing the mechanism of PARL cleavage, PARLase

discovery may shed light on the structural characteristics that PARL substrates must hâve in

order to be cleaved by this unique rhomboid protease.

PARL in rétrograde régulation

Rétrograde régulation is a new emerging field of mitochondrial research that involves the rôle of

this organelle in intracellular signalling (Butow & Avadhani 2004). Implicit in this définition is

21

that mitochondrial signaling occurs in the opposite direction to that of the more familiar

anterograde régulation characterized by the transfer of information and material from the nucleus

and cytoplasm to mitochondria.

It is now well established that cells modulate nuclear gène expression in response to

mitochondrial activities and dysfunctions. For instance, impaired mitochondrial oxidative

phosphorylation or DNA loss (pO), activâtes the Rtgl/Rtg3 transcription factor complex, thereby

triggering anapleurotic reactions to maintain cellular glutamate levels in the absence of full TCA

cycle activity. Inhibition of électron transport by antimycin A as well as dissipation of the

mitochondrial membrane potential by CCCP were also found to resuit in the repression of gènes

(Epstein et al 2001, Hughes et al 2000). Thus, the définition of rétrograde régulation includes

nuclear responses to changes in the functional state of mitochondria.

How mitochondria control nuclear activities? Pioneering studies in the yeast Saccharomyces

cerevisiae hâve shown that peptides extruded from the mitochondria participate in the régulation

of nuclear gènes (Arnold et al 2006, Traven et al 2001). The first évidence for a release of

peptides from mitochondria came from the observation that peptides derived from

mitochondrially encoded proteins were detected at the cell surface of mammalian cells in

association with major histocompatibility antigen class I molécules (Loveland et al 1990). Today,

over 270 peptides, ranging in size between 6 and 26 amino acids, hâve been identified to be

continuously exported from the matrix to the cytosol in an ATP- and temperature-dependent

manner (Augustin et al 2005). They resuit from the catabolism of over 40 matrix and IMM

proteins. Recently, élégant genetic studies hâve shown that deletion of the yeast YME1 gène,

which encodes the inner membrane i-AAA protease, abolished a large number of thèse peptides

and led to the induction of nuclear gènes with functions in mitochondrial gène expression and the

biogenesis of the respiratory chain (Arnold et al 2006). This suggests that peptides released from

the mitochondria exécute a mitochondria-to-nucleus signalling pathway, which, in respiring cells,

allows the re-adjustment of the biogenesis of the respiratory chain in response to an altered

activity of the FjFo-ATP synthase.

PARL is emerging as a central regulator of major mitochondrial activities. It is conceivable to

speculate, therefore, that this rhomboid protease participâtes in rétrograde régulation. Data

supporting this possibility hâve recently started to émerge. We hâve shown that PARL p-

22

cleavage releases in the matrix a 25 amino acid-long peptide which, in transfection studies, is

efficiently targeted to the nucleus (Sik et al 2004). Whether this peptide, termed Pp, is exported

from the matrix to the cytosol remains to be demonstrated. However, data indicating the présence

of the Pp peptide in the nucleus of undifferentiated neurons (our unpublished data) and the

existence of a specialized machinery for the export of matrix peptides of similar size strengthen

this notion (Augustin et al 2005). Participation of the Pp peptide in rétrograde régulation would

bring PARL within the mainstream concept of RIP, where a signalling protein is subjected to

highly regulated release. Unlike other I-CliPs, however, the case of PARL would be the fïrst

example of RIP where the putative signaling moiety is part of the I-CliP itself. Given the major

rôle of PARL in mitochondria biology and the far-reaching implications of rétrograde régulation

in development, aging, disease, and environmental adaptation, the study of Pp signaling is

anticipated to lead to major advances in our understanding of cell biology and mechanisms of

mitochondrial diseases.

23

Material and methods

DNA préparation

Plasmid DNA préparations can be done by miniprep or PEGprep. Miniprep allows us to purify

small quantities of DNA ( 5 - 1 0 ug) from small volumes (1-2 ml) of bacterial culture in about 30

minutes. This DNA can be used for transformation and cloning purposes. PEGprep allows us to

obtain transfection grade plasmid in large quantities (~5 mg) from 250 ml of bacterial culture.

DNA miniprep

Plasmid minipreps were done using the high pure plasmid isolation kit from Roche applied

science. This procédure is used to extract plasmid DNA from bacterial cell suspensions and is

based on the alkaline lysis procédure.. The procédure takes advantage of the fact that plasmids

are relatively small supercoiled DNA molécules and bacterial chromosomal DNA is much larger

and less supercoiled. This différence in topology allows for sélective précipitation of the

chromosomal DNA and cellular proteins from plasmids and RNA molécules. The cells are lysed

under alkaline conditions, which dénature both nucleic acids and proteins, and when the solution

is neutralized by the addition of potassium acétate, chromosomal DNA and proteins precipitate

because it is impossible for them to renature correctly owing to their large size. Plasmids on the

other hand, renature and stay in solution, effectively separating them from chromosomal DNA

and proteins. The process does not require DNA précipitation, organic solvent extractions, or

extensive handling of the DNA.

Protocol: Grow bacterial cultures overnight in a rotary shaker at 200 rpm and 37° C in LB

médium (lOg/L Tryptone, 5g/L Yeast extract, and lOg/L NaCl) with the required antibiotic based

on the vector used. Centrifuge the cells at 13000 rpm for 2 minutes. Resuspend the pellet in 250

ul of suspension buffer containing RNase A followed by the addition of 250 (il of lysis buffer.

Invert the tube 2 or 3 times to facilitate even lysis of cells in the suspension. In about 2 minutes,

add 350 ul of neutralizing solution followed by gentle inversions of the tube. Place the tube in ice

for 10 minutes followed by centrifugation for 10 minutes at 13000 rpm. Remove the supernatant

carefully and transfer to the purification column followed by a minute of centrifugation. Discard

the flow through and add 750 ul of wash buffer followed by two centrifugations where the flow

24

through has to be discarded in both. Purified plasmid DNA can be eluted by adding 50 jul of

elution buffer (TE; 10 mM Tris, 1 mM EDTA) followed by centrifugation at maximal speed for a

minute.

DNA maxiprep for cell transfection (PEG prep)

Protocol: Incubate a single bactcrial colony in 150 ml of Terrifie Broth with the appropriate

antibiotic at 37°C in a rotary shaker at 135 rpm overnight. Centrifuge at 4000 rpm in swing

bucket rotor for 10 minutes. Resuspend pellet in 4ml of Solution I (50mM glucose, 25mM Tris-

HCL pH 7.6, lOmM EDTA), transfer to corning tube and add 5 ul of RNase A (lOmg/ml,

Fermentas). Add 8 ml of Solution II (0.2M NaOH and 1%SDS). Invert gently 2-3 times and add

4ml of Solution III (3M KAc, 11% v/v glacial acetic acid). Invert gently 2-3 times and place in

ice for 10 minutes. Centrifuge for 10 minutes at 4°C 14000 rpm and if supernatant is still turbid,

transfer to a new tube and recentrifuge. Transfer the clear supernatant to a new tube and note the

volume. Add 0.6 volumes of Isopropanol followed by inversions of the tube and incubation at -

20°C for an hour. Centrifuge at 14000 rpm at 4°C for 20 minutes. Discard supernatant. Air dry

the pellet for 5 minutes and resuspend it in 600 ul of TE (lOmM Tris-HCL pH 7.6 and lmM

EDTA) and transfer to an eppendorf tube. Add 3 ul of RNase A and incubate for 30 minutes at

37°C. Centrifuge at full speed for 10 minutes and transfer the supernatant to a new eppendorf

tube and note the volume. Add an equal volume of PEG (20% PEG and 2.5M NaCl) and invert

the tube few times. Incubate in ice for 30 minutes.

Centrifuge for at 14000 rpm for 10 minutes and resuspend pellet in 500 ul of TE, add one volume

of phenol/chloroform (Phenol:Chloroform:Isoamyl alcohol 25:24:1 saturated with lOmM Tris pH

8.0 and lmM EDTA; Sigma), vortex for 10 seconds, centrifuge for 5 minutes at 14000 rpm and

collect the upper layer and note the quantity. Repeat the above phenol/chloroform step once again

with the collected upper layer. Collect the upper layer again and add an equal volume of

chloroform (Sigma), vortex for 30 seconds, and centrifuge at 14000 rpm for 5 minutes and collect

the supernatant. Add 0.1 volume of Na Acétate pH 5.3 followed by three volumes of 100%

cthanol and invert the tubes a few times. Centrifuge at full speed for 10 minutes and discard the

supernatant. Add 600 ul of 70% ethanol and centrifuge for 10 minutes at 14000 rpm and discard

the supernatant. Resuspend the pellet in 1 ml of TE. Quantify the DNA by measuring the optical

density at 260 nm.

25

Mutagenesis

Mutagenesis was performed based on the oligonucleotide based site-directed mutagenesis

methodology. In this strategy, two primers are used. A mutagenic primer that introduces the

desired mutation and a sélection primer containing a mutation in the récognition séquence for a

unique restriction enzyme site. The two primers simultaneously anneal to one strand of the

denatured double-stranded plasmid under conditions favoring the formation of hybrids between

the primers and the DNA template. After standard DNA elongation, ligation, and a primary

sélection by restriction digest, the mixture of mutated and unmutated plasmids is transformed into

a mutS E. coli strain defective in mismatch repair. Transformants are pooled, and plasmid DNA

is prepared from the mixed bacterial population. The isolated DNA is then subjected to a second

sélective restriction enzyme digestion. Since the mutated DNA lacks the restriction enzyme

récognition site, it is résistant to digestion. The parental DNA, however, is sensitive to digestion

and will be linearized, rendering it at least 100 times less efficient in transformation of bacterial

cells. A final transformation with the selectively digested DNA results in highly efficient and

spécifie recovery of the desired mutated plasmid.

The advantages of this strategy are as follows:

> Does not require single-stranded vectors or specialized double-stranded plasmids.

> Does not require viral transductions.

> Does not require subcloning.

> Results in mutation efficiencies of >70-90%.

In addition, the use of T4 DNA polymerase instead of PCR reduces the risk of introducing

spurious mutations.

The following figure (Fig 5.1) explains schematically the entire procédure.

26

UniqueRestriction •Site

1. Dénature dsDNA

2. Re-anneal with Pnmers

3. Synthesize second strandwith T4 DNA Polymerase andseal gaps with T4 DNA Ligase;primary digestion with sélectionrestriction enzyme.

4. Transform mutS E. coliFIRST TRANSFORMATION

5. Isolate DNA froin transformant pool

i i Target Vi | Parental\ \ Plasmid Clonedgene

SELECTIONPRIMER

MUTAGENICPRIMER

" \ \

6 Secondary digestion withsélection enzyme

7. Transform E coliSECOND (FINAL) TRANSFORMATION

8 Isolate DNA fromindividual transformantsto confirm présence ofdesired mutation

Fig 5.1 Scheme of the mutagenesis protocol.

27

Primers design

Sélection Primer: The fonction of the sélection primer is to eliminate the original unique

restriction enzyme site. The sélection primer can be designed by incorporating one or more base

pair changes within the targeted unique restriction site. Since restriction enzymes recognize an

exact DNA séquence, one or more base pair change(s) within the récognition séquence should be

suffïcient to abolish the restriction digestion. If the sélection restriction site is located within a

gène, avoid using a

sélection primer that will introduce change that could interfère with the expression of that gène

(e.g., by causing a reading-frame shift or a prématuré termination codon).

Mutagenic Primers: The function of the mutagenic primer is to introduce the desired mutation. In

our experiments on phosphorylation studies, we substituted phosphorylable amino acids (Serine,

Threonine and Tyrosine) with amino acids that either mimick phosphorylation (Asp; (Lee et al

2004a)) or are unphosphorylable (Ala).

Important:

a) The mutagenic and sélection primers must anneal to the same strand of the plasmid.

b) The distance between the sélection primer and the mutagenic primer is not critical. If

possible, however, design the sélection and mutagenic primers so that they will be evenly

spaced after annealing to the DNA template; this will allow the DNA polymerase to

extend both primers an équivalent distance. In rare cases, where the unique restriction site

and the targeted mutagenic site are very close to each other, one single primer can be

designed to introduce both mutations simultaneously.

c) In most cases, 10 nucleotides of uninterrupted matched séquences on both ends of the

primer (flanking the mismatch site) should give suffïcient annealing stability, provided

that the GC content of the primer is greater than 50%. If the GC content is less than 50%,

the lengths of

d) the primer arms should be extended accordingly. The mismatch bases should be placed in

the center of the primer séquence. For optimum primer annealing, the oligonucleotides

should start and end with a G or C. The annealing strength of the mutagenic primer

should always be

e) equal to or greater than that of the sélection primer.

28

f) There can be more than one base mismatch in the mutagcnic primer. However, the

success rate for targeted mutagenesis of a plasmid is optimized when there are only one or

a few mismatches in the primer

Primers phosphorvlation

The primers must be phosphorylated at the 5' end, so that they can bc ligated to the 3' end of the

newly synthesized strand.

Protocol: Resuspend primers in water at a concentration of 1 ug/ul. For each primer prépare a

reaction mix of 20 ul (final volume) eomposed of 2 JLXI of lOmM ATP (Fermentas), 2 ul of 10X

PNK Buffer A (500 mM Tris-HCL pH 7.6, 100 mM MgCl2, 50 mM DTT, 1 mM Spermidine, 1

mM EDTA), 1 ul of primer, and 1 \û of T4 Polynucleotide Kinase (PNK). Incubate for 30

minutes at 37° C and inactivate PNK and 75° C for 10 minutes. Briefly spin the tubes after this

step.

Plasmid denaturation and primers annealing

Protocol: To a final volume of 30 ul, add 1 ul of the phosphorylated sélection primer, 1 ul of the

phosphorylated mutagenic primer, 1 ul of the DNA template (100 ng/ul concentration), 3 ul of

Fermenta's Tango 10X buffer (330 mM Tris-acetate pH 7.9, lOOmM magnésium acétate, 66 mM

Potassium acétate, lmg/ml BSA). Incubate at 100 ° C for 3 minutes and immediately place in ice

for 5 minutes.

Synthesis of the mutant DNA strand and restriction digestion sélection

Protocol: To each tube of the the primer/plasmid annealed mix, add 1 ul of T4 DNA Ligase (10

U/ul; Fermentas), 1 ul of T4 DNA Polymerase (10 U/ ul, Fermentas), 1 ul of dNTP and ATP mix

(2.5 mM dNTP and 1 mM ATP). Mix well and centrifuge briefly. Incubate at 37°C for 60

minutes. Stop the reaction by heating at 75°C for 10 minutes to inactivate the enzymes. After

allowing cooling, add 1 ul of the required restriction enzyme to digest the unmodified plasmids

and incubate at 37°C for 60 minutes.

29

Transformation into MutS cells

The purpose of this step is to amplify the mutated strand (as well as the parental strand) in heat

shock-competent mutS (repair-deficient) E. coli cells (BMH 71-18 MutS; Clontech or Promega)

Protocol: Preheat a water bath to 42°C. Add 2 jal of the digested plasmid DNA digestion (see

above) to 50 ul of MutS cells and incubate on ice for 10 minutes. Heat shock cells of 42°C for 40

seconds, incubate in ice for 2 minutes, and add 500 ul of LB média. Incubate at 37°C for 45

minutes and add to a Falcon tube containing 5 ml of LB média with the required antibiotic. Shake

overnight at 37°C at 200 rpm.

Isolation of the mixed plasmid pool by miniprep

Protocol: Do a DNA miniprep from 2 ml of E.coli MutS cells.

Sélection of the Mutant Plasmid

This procédure sélects for the mutant plasmid by complète digestion of parental (unmutated)

plasmids.

Protocol: Digest 15 (xl of the miniprep done above (~1 ug) in 20 ul final volume; transform 2 ul

in 50 ul of compétent E.coli K12 cells; plate 1/10 of the transformed cells in LB agar média

containing the appropriate antibiotic.

Mutants screening

Mutants are identifïed by sequencing. In some cases the mutagenic primer can be designed in

such a way that along with the mutation, a restriction site can be introduced or eliminated. In such

cases, restriction digestion should be performed and potential mutants analysed by agarose gel

electrophoresis.

DNA sequencing

Plasmid sequencing was done using the sequencing service from Université Laval.

Chromatogram files were analysed using Chromas Pro from Technelysium Ltd.

30

Mammalian cell culture and transfection

HEK 293, HeLa and Cos-7 cells were obtained from American Type Culture Collection (ATCC).

DMEM (Dulbecco's Modified Eagle Médium, Invitrogen) was used for HEK 293 and Cos-7 cells

while MEM (Minimum Essential Médium, Sigma) was used for HeLa cells. In ail cases, the

média was supplemented with 10% fetal bovine sérum, 2mM glutamine, 10 jo.g/jj.1 penicillin and

10 jag/jj.1 streptomycin. Maintain cells in culture by growing them in pétri dishes. When the cells

are 100% confluent, remove and discard culture médium. Briefly rinse the cell layer with 0.25%

(w/v) Trypsin- 0.53 mM EDTA solution to remove ail traces of sérum that contains trypsin

inhibitor. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an

inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid

dumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to

detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersai. Add 6.0 to

8.0 ml of complète growth médium and aspirate cells by gently pipetting. Add appropriate

aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Médium renewal: Every 2 to 3 days

For transfection, grow cells in 6 well plates until they reach 50% of confluence.

Per every well of cells to transfect, add 1 jug of DNA (PEG prep) to 100 ul of sérum free média

and add 3 \A of Fugene 6 (Roche Applied Science). Incubate for 30 minutes and add the mix to

the cells.

To check transfection efficiency, every experiment included a control transfection with a plasmid

expressing GFP (pEGFP-Nl, Clontech).

Cell harvesting

Protocol: When transfected cells reach 90-100% of confluence (24-48 hours after transfection,

depending on the cell type), add 800 ul of RIPA (65 mM tris base, 0.15 M NaCl, 1% NP40,

0.25% sodium deoxycholate, lmM EDTA pH 7.4) to each well. Centrifuge cell lysate at 14000

rpm at 4°C for 30 minutes. Collect the supernatant and continue with immunoprecipitation or

freeze at -30°C for further use.

31

1. Lyse cells, addaffinity resin

2. Incubate to allowcomplex formation

EJPf 3. Spin

Immunoprecipitation

Protocol: Wash the resin with covalently attached antibody (anti-

FLAG or anti-HA or anti-PARL C Term) thrice in IX STEN-

NaCl (50 mM Tris pH 7.6, 500 mM NaCl, 2 mM EDTA, 0.2%

NP40) and add 15 u.1 of the slurry to 800 ul of cell lysate; allow

mixing by gentle rotation at 4°C overnight (Fig 5.2). Wash the

beads thrice with IX STEN-SDS (50 mM Tris pH 7.6, 500 mM

NaCl, 2 mM EDTA, 0.2% NP40, 0.1% SDS), leaving at the end

the beads in 50 ul of buffer.

Fig 5.2, Scheme showing immunoprecipitation

Migration of proteins in gel electrophoresis

Protocol: Prépare IX SDS Running Buffer by adding 50 ml 20X

NuPAGE (Invitrogen) MES or MOPS SDS running buffer to 950

ml of deionized water. Prépare the electrophoresis setup and

install the precast 4-12% Bis Tris gel and add the running buffer.

Add the appropriate amount of NuPAGE LDS 4X buffer (Invitrogen) and DTT (1M, 10X) and

incubate at 85°C for 4 minutes. During this incubation period, remove the comb from the gel and

wash the gel pockets with the running buffer.

Add the appropriate amount of sample (~12 fxl) per well. Add 3 ul of Seeblue Plus Prestained

Ladder (Invitrogen) to one of the well s (usually the first or the last). Attach electrical supply and

run at 200 Volts until the blue dye front reaches the bottom of the gel.

4. Wash

5. Elute

Protein of Interest•» Other ComponentsS Affinity Resin

Western Blotting

Protocol: Prépare 1 liter of IX NuPAGE Transfer Buffer by adding 50 ml 20X NuPAGE

Transfer Buffer and 100 ml methanol to 850 ml deionized water. Soak blotting pads in 700 ml of

1X NuPAGE Transfer Buffer. Prépare transfer membranes by moistening in methanol and then

immediately adding transfer buffer. Soak the filter paper briefly in Transfer Buffer. Place a pièce

of pre-soaked filter paper on top of the gel (adhered to the bottom plate) and remove any trapped

air bubbles. Turn the plate over so the gel and filter paper are facing downwards over a gloved

32

hand or clean flat surface. Place a pre-soaked transfer membrane on the gel and remove trapped

air bubbles. Place another pre-soaked filter paper on top of the membrane and remove any air

bubbles. Place two soaked blotting pads into the cathode (-) core of the blot module. Carefully

pick up the gel/membrane assembly and place on blotting pad in the correct orientation, so the gel

is closest to the cathode core. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of

cathode core. Place the anode (+) core on top of the pads. Hold the blot module together fïrmly

and slide it into the guide rails on the lower buffer chamber. Insert the Gel Tension Wedge into

the Lower Buffer Chamber and lock the Wedge into position. Fill the blot module with Transfer

Buffer prepared until the gel/membrane assembly is covered. Fill the Outer Buffer Chamber with

650 ml deionized water. Place the lid on the unit and connect the electrical leads to the power

supply. Perform transfer for nitrocellulose or PVDF membranes using 100 V constant for 1 hour.

Remove the cassette and rinse the membrane with PBST (137mM NaCl, 27 mM KC1, 43 mM

Na2HPO4, 14 mM KH2PO4, 1% Tween-20). Proceed with blocking the membrane with 5% milk

solution made in PBST. Add the required antibody at its optimal dilution and incubate. The

following table gives the dilution and concentration of the various antibodies used:

Primary Antibody

Anti-Flag Rabbit

Anti-Flag Mouse

Anti-PARL N-Term

Anti- PARL C-Term

Anti- Myc Rabbit

Anti-GFP Mouse

Dilution

1:1000

1:1000

1:1000

1:1000

1:1000

1:10000

Incubation time

1 hour, room température

1 hour, room température

2 hours, room température

Overnight 4°C

Overnight 4°C

2 hours, room température

After the incubation with the primary antibody, wash the membranes with PBST thrice and add

HRP conjugated secondary antibody (1:2000 dilution; Chemicon). Incubate at room température

for 1 hr and wash for 15 minutes with PBST, changing washing solution from time to time.

Reveal blotted proteins by chemiluminescence with Super Signal West Femto (Pierce) using the

Versadoc imaging station (Biorad).

33

Other antibodies used during the course of the study were: polyclonal anti-GFP (Clontech,

Mountain View, CA), monoclonal anti-GFP (Invitrogen, Carlsbad, CA), monoclonal anti-

cytochrome c and monoclonal anti-HA (Covance, Denver, PA), mouse M2 monoclonal and

rabbit polyclonal anti-FLAG.

Anti-PARL antibodies

For the préparation of the anti-PARL-N-Term antibody (directed against a PARL peptide located

near the N terminus), a 12-amino acid-long peptide spanning amino acids 54-66 of PARL

(54RKAPRKVEPRRSD66) was synthesized, purified by high pressure liquid chromatography,

conjugated to bovine sérum albumin, and used to immunize New Zealand rabbits according to

standard 90-day protocols for antisera production (Covance). Antiserum (bleed 2 and 3) was used

and diluted 1:1000 and 1:100 for immunoblotting and immunoprecipitation, respectively. The

antibody used for immunocytochemical analysis was obtained by peptide affïnity-purification of

the antisera and used 1:2000 (1:200 in immunoblot). The specificity of the anti-PNT

immunostaining was addressed by pre-adsorbing the antisera with a recombinant GST-PARL N-

terminal fusion protein. Anti-PNT immunostaining was abolished when the antibody was

incubated 1-4 h at 4°C with lysate of Escherichia coli cells expressing a recombinant GST-

PARL N-terminal (amino acid 20-96 of PARL) fusion protein.

Anti-PARL-C-Term was raised against a peptide spanning the last 21 amino acids of PARL

(359PLVKIWHEIRTNGPKKGGGSK379) conjugated to the N-terminus of keyhole limpet

hemocyanin; antisera was collected according to standard immunization protocols. Among the 6

bleeds tested, bleed n.3 best detected PARL in immunoprecipitation (used at 1:100 dilution) and

immunoblot analysis (used at 1:1000 dilution).

Antibody specificity was tested by preadsorbing the antisera with 1 ng of the peptide used to

immunize the rabbits.

Crosslinking of anti-PARL-C-Term to protein A agarose beads

Covalent coupling of the anti-PARL-C-Term was performed using the SeizeX

immunoprecipitation kit from Pierce. This kit offers an improvement over the classical method of

immunoprecipitation by immobilizing the antibody to the Protein A support using the cross-

34

linker DSS (disuccinimidyl suberate). This procédure results in a permanent affinity support with

a properly oriented antibody. The crude sample is then incubated with the immobilized antibody

to form the immune complex. The affinity support is washed by centrifugation using a Spin Cup

Column and the remaining antigen is dissociated from the antibody using an elution buffer. The

primary antibody does not contaminate the final antigen préparation and the immobilized

antibody support is preserved for future IPs. Depending on the stability of the immobilized

antibody, the prepared affinity support may be used 2-10 times, thus conserving precious

antibody.

Since the antibody concentration in the antisera may vary, covalent coupling should be tested

using various antisera dilutions, so that optimal chemical crosslinking efficiency can be obtained

for immuoprecipitation. Among the dilutions tested, (1:25, 1:50, 1:100, 1:200, 1:500) bleed-3

diluted 1:100 gave us the best results.

Protocol: Equilibrate the Immobilized Protein A and reagents to room température. Add 500 ml

of ultrapure water to the dry-blend buffer. To store excess buffer, add a preservativc such as

0.02% sodium azide and store at 4°C. Gently swirl the bottle of ImmunoPure Immobilized

Protein A to obtain an even suspension. Add 0.4 ml of the Immobilized Protein A (50% slurry)

into one of the Spin Cup Columns and place inside a Microcentrifuge Tube and centrifuge (Note:

Ail centrifugations in this procédure should be perfomed at 4000 rpm and at room température

for 1 minute). Discard flow-through and replace spin cup into the tube. Wash gel by adding 0.4

ml of binding/wash Buffer to the gel. Cap tube and resuspend gel by inverting with gentle

shaking. Centrifuge the tube and discard flow-through and replace spin cup into tube. Repeat this

step once. Place spin cup into a new microcentrifuge tube. Apply 50-500 ug of purified antibody

prepared in 0.3-0.4 ml of binding/wash Buffer to the prepared gel. Cap the microcentrifuge tube

and place it on a rocker for at least 15 minutes to allow the antibody to bind to the gel. Centrifuge

the tube. If desired, save the flow-through to estimate the amount of antibody bound to the

Protein A. Place spin cup into another microcentrifuge tube and add 0.5 ml of binding/wash

Buffer. Invert tube 5-10 times. Centrifuge tube and discard flow-through. Repeat this step two

additional times using the same collection tube. Transfer the spin cup into a new microcentrifuge

tube and add 0.4 ml of binding/wash Buffer.

35

Puncture the foil covering of a single tube of No-Weigh™ DSS with a pipette tip and add 80 ul

of DMSO or DMF. Use the pipette to thoroughly mix the solution (Le., draw up and expel the

solution) until the DSS is dissolved. Add 25 ul of the DSS solution to the spin cup eontaining the

bound antibody support. The tube eontaining the reconstituted DSS can be discarded from the

strip by pushing tube from the bottom, away from the strip. Note: Because DSS is a hydrophobic

molécule, a microprecipitate may form when it is added to the aqueous médium, which results in

a cloudy appearance. Nevertheless, the reaction will proceed efficiently and the microprecipitate

may disappear during conjugation. Place the top cap on the tube and gently mix for 30-60

minutes. Centrifuge tube and discard flow-through. Add 500 ul of elution buffer (the elution

buffer (pH 2.8) should be used with care since excessive time in the elution buffer was observed

to disrupt the covalent coupling.) to the spin cup. Cap tube and invert it 10 times. Centrifuge tube

and discard flow-through. Place the spin cup back into the microcentrifuge tube. Note: The pH of

the elution buffer is 2.8 and will elute IgG that is not covalently coupled to the Immobilized

Protein A. The majority of polyclonal antibodies and most monoclonal antibodies can tolerate

low pH conditions for short durations. Repeat this four additional times to quench the reaction

and to remove excess DSS and uncoupled antibody. Place the spin cup in a new microcentrifuge

tube and wash gel two times using 500 ul Binding/Wash Buffer.

Isolation of endogenous PARL from human placental mitochondria

Protocol: Thaw frozen human placental mitochondria (~ 250 mg) in ice and centrifuge at 14000

rpm at 4°C for 10 minutes. Remove the supernatant and resuspend the pelleted mitochondria in

500 ul of IX PBS and add 500 ul of 2X STEN-SDS with protease inhibitors. Gently mix at room

température for 10 minutes. Add 40 ul of beads slurry on which the anti PARL-C-Term has been

covalently coupled (see above protocol). Mix gently overnight at 4°C, wash the beads thrice in

IX STEN-SDS and proceed with the western blotting analysis.

36

Purification of endogenous and transfected PARL

Immunoprecipitated endogenous or transfected PARL was purified by gel electrophoresis. After

the run, gels were stained overnight with colloidal coomassie blue (GelCode; Pierce) at 4°C.

Destaining of the gel was done by repeated washes in distilled water. Bands corresponding to the

mature mitochondrial form of PARL (MAMP) were excised, frozen at -80 °C and sent to 21st

Century Biochemicals for mass spectrometric analysis.

Mass Spectrometry

Mass spectrometric analysis was performed by employing the services of 21st Century

Biochemicals. However, ail the différent PARL proteins analyzed by this method were purified in

Dr. Pellegrini's laboratory. See published work (Jeyaraju et al 2006) for détails concerning this

methodology.

Mitochondria Morphology analysis

Mitochondria morphology and PARL topology analysis were done by the McBride lab. However,

ail constructs used were generated in Dr. Pellegrini's laboratory. See published work (Jeyaraju et

al 2006) for détails concerning this methodology.

Cloning

For PCR based cloning, KOD hot start DNA polymerase (Novagen) was used following

manufacurer's instructions. In ail cases, linearized and dephosphorylated vectors were ligated to

the phosphorylated PCR product and positive clones were identified by PCR screening DNA

sequencing. Standard cloning procédures were used throughout the course of this study.

Vectors

The two mammalian expression vectors used in this study are pcDNA3 (for expression of

FLAG/HA/cMyc-tagged proteins; Invitrogen), and pEGFPNl (for expression of GFP tagged

proteins; Novagen).

37

Références bibliographiques

Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. 2002. Three-dimensional structure of theapoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell9: 423-32

Adrain C, Slee EA, Harte MT, Martin SJ. 1999. Régulation of apoptotic protease activating factor-1oligomerization and apoptosis by the WD-40 repeat région. J Biol Chem 21 A: 20855-60

Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, et al. 2000. OPA1, encoding a dynamin-relatedGTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nui. Genêt. 26:211-5

Alirol E, James D, Huber D, Marchetto A, Vergani L, et al. 2006. The Mitochondrial Fission Protein hFislRequires the Endoplasmic Reticulum Gateway to Induce Apoptosis. Mol Biol Cell

Ankarcrona M, Hultenby K. 2002. Presenilin-1 is located in rat mitochondria. Biochem Biophys Res Commun295: 766-70

Arnold I, Wagner-Ecker M, Ansorge W, Langer T. 2006. Evidence for a novel mitochondria-to-nucleussignalling pathway in respiring cells lacking i-AAA protease and the ABC-transporter Mdll. Gène367: 74-88

Augustin S, Nolden M, Muller S, Hardt O, Arnold I, Langer T. 2005. Characterization of peptides releasedfrom mitochondria: évidence for constant proteolysis and peptide efflux. J Biol Chem 280: 2691-9

Ben-Shem A, Fass, D. & Bibi, E. Proc. Natl. Acad. Sci. USA (in thepress).Bier E, Jan LY, Jan YN. 1990. rhomboid, a gène required for dorsoventral axis establishment and peripheral

nervous System development in Drosophila melanogaster. Gènes Dev 4: 190-203Brown M, Goldstein J. 1999. A proteolytic pathway that controls the cholestérol content of membranes, cells,

and blood. Proc Natl Acad Sci USA96: 11041-8Brown MS, Ye J, Rawson RB, Goldstein JL. 2000. Regulated intramembrane proteolysis: a control

mechanism conserved from bacteria to humans. Cell 100: 391-8Butow RA, Avadhani NG. 2004. Mitochondrial signaling: the rétrograde response. Mol Cell 14: 1-15Chan D, Frank S, Rojo M. 2006. Mitochondrial dynamics in cell life and death. Cell Death DifferChen H, Chan DC. 2005. Emerging functions of mammalian mitochondrial fusion and fission. Hum Mol Genêt

14 Spec No. 2: R283-9Chen H, Chomyn A, Chan DC. 2005. Disruption of fusion results in mitochondrial heterogeneity and

dysfunction. J Biol Chem 280: 26185-92. Epub 2005 May 17.Chen H, Detmer SA, Ewald AJ, Griffln EE, Fraser SE, Chan DC. 2003. Mitofusins Mfnl and Mfn2

coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol160: 189-200

Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L. 2004. OPA1 requires mitofusin 1 to promotemitochondrial fusion. Proc Natl Acad Sci USA 101: 15927-32. Epub 2004 Oct 27.

Cipolat S, Rudka T, Hartmann D, Costa V, Serneels L, et al. 2006. Mitochondrial rhomboid PARL régulâtescytochrome c release during apoptosis via OPAl-dependent cristae remodeling. Cell 126: 163-75

De Strooper B, Annaert W, Cupers P, Saftig P, Craessaerts K, et al. 1999. A presenilin-1-dépendent gamma-secretase-like protease médiates release of Notch intracellular domain. Nature 398: 518-22

De Strooper B, Saftig P, Craessaerts K, Vanderstichele H, Guhde G, et al. 1998. Deficiency of presenilin-1inhibits the normal cleavage of amyloid precursor protein. Nature 391: 387-90

Delettre C, Griffoin JM, Kaplan J, Dollfus H, Lorenz B, et al. 2001. Mutation spectrum and splicing variantsin the OPA1 gène. Hum Genêt 109: 584-91.

Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, et al. 2000. Nuclear gène OPA1, encoding amitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genêt 26: 207-10

Dimmer KS, Fritz S, Fuchs F, Messerschmitt M, Weinbach N, et al. 2002. Genetic Basis of MitochondrialFunction and Morphology in Saccharomyces cerevisiae. Mol Biol Cell 13: 847-53.

Epstein CB, Waddle JA, Haie Wt, Dave V, Thornton J, et al. 2001. Genome-wide responses to mitochondrialdysfunction. Mol Biol Cell 12: 297-308

Esser K, Tursun B, Ingenhoven M, Michaelis G, Pratje E. 2002. A novel two-step mechanism for removal of amitochondrial signal séquence involves the mAAA complex and the putative rhomboid protease Pcpl.J Mol Biol 323: 835-43

38

Forte M, Bernardi P. 2006. The permeability transition and BCL-2 family proteins in apoptosis: co-conspirators or independent agents? Cell Death Differ 13: 1287-90

Frank S, Gaume B, Bergmann-Leitner ES, Leitner WW, Robert EG, et al. 2001. The rôle of dynamin-relatedprotein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell 1: 515-25

Frey TG, Mannella CA. 2000. The internai structure of mitochondria. Trends Biochem. Sci. 25: 319-24Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, et al. 2006. OPA1 controls apoptotic

cristae remodeling independently from mitochondrial fusion. Cell 126: 177-89Guan K, Farh L, Marshall TK, Deschcnes RJ. 1993. Normal mitochondrial structure and génome

maintenance in yeast requires the dynamin-like product of the MGM1 gène. Curr Genêt 24: 141-8.Gupta S, Singh R, Datta P, Zhang Z, Orr C, et al. 2004. The C-terminal tail of presenilin régulâtes Omi/HtrA2

protease activity. J Biol Chem 279: 45844-54Harris RA, Hawes JW, Popov KM, Zhao Y, Shimomura Y, et al. 1997. Studies on the régulation of the

mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases. Adv Enzyme Regul 37:271-93

Hcrlan M, Bornhovd C, Hell K, Neupert W, Reichert AS. 2004. Alternative topogcnesis of Mgml andmitochondrial morphology dépend on ATP and a functional import motor. J Cell Biol 165: 167-73.Epub 2004 Apr 19.

Hcrlan M, Vogel F, Bornhovd C, Neupert W, Reichert AS. 2003. Processing of Mgml by the Rhomboid-typeprotease Pcpl is required for maintenance of mitochondrial morphology and of mitochondrial DNA.J Biol Chem

Horbinski C, Chu CT. 2005. Kinase signaling cascades in the mitochondrion: a matter of life or death. FreeRadie Biol Med 38: 2-11

Hughes TR, Marton MJ, Jones AR, Roberts CJ, Stoughton R, et al. 2000. Functional discovery via acompendium of expression profiles. Cell 102: 109-26

Ingerman E, Perkins EM, Marino M, Mears JA, McCaffery JM, et al. 2005. Dnml forms spirals that arestructurally tailored to fit mitochondria. J Cell Biol 170: 1021-7

Ishihara N, Fujita Y, Oka T, Mihara K. 2006. Régulation of mitochondrial morphology through proteolyticcleavage of OPA1. Embo J 25: 2966-77

Jeyaraju DV, Xu L, Letellier MC, Bandaru S, Zunino R, et al. 2006. Phosphorylation and cleavage ofpresenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology.Proc Natl Acad Sci U S A 103: 18562-7

Karbowski M, Arnoult D, Chen H, Chan DC, Smith CL, Youle RJ. 2004. Quantitation of mitochondrialdynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked duringthe Bax activation phase of apoptosis. J Cell Biol 164: 493-9. Epub 2004 Feb 09.

Karbowski M, Youle RJ. 2003. Dynamics of mitochondrial morphology in healthy cclls and during apoptosis.Cell Death Differ 10: 870-80.

Koch A, Yoon Y, Bonekamp NA, McNiven MA, Schrader M. 2005. A rôle for Fisl in both mitochondrial andperoxisomal fission in mammalian cclls. Mol Biol Cell 16: 5077-86

Koonin EV, Makarova KS, Rogozin IB, Davidovic L, Letellier MC, Pellegrini L. 2003. The rhomboids: anearly ubiquitous family of inIramembrane serine proteases that probably evolved by multipleancient horizontal gène transfers. Génome Biol 4: R19

Koshiba T, Detmer SA, Kaiser JT, Chen H, McCaffery JM, Chan DC. 2004. Structural basis of mitochondrialtethering by mitofusin complexes. Science 305: 858-62.

Kuroiwa T, Nishida K, Yoshida Y, Fujiwara T, Mori T, et al. 2006. Structure, function and évolution of themitochondrial division apparatus. Biochim Biophys Acta 1763: 510-21

Lee SY, Wenk MR, Kim Y, Nairn AC, De Camilli P. 2004a. Régulation of synaptojanin 1 by cyclin-dependentkinase 5 at synapses. Proc Natl Acad Sci USA 101: 546-51

Lee YJ, Jeong SY, Karbowski M, Smith CL, Youle RJ. 2004b. Rôles of the mammalian mitochondrial fissionand fusion mediators Fisl, Drpl, and Opal in apoptosis. Mol Biol Cell 15: 5001-11

Liu X, Kim CN, Yang J, Jemmerson R, Wang X. 1996. Induction of apoptotic program in cell-free extracts:requirement for dATP and cytochrome c. Cell 86: 147-57

Loveland B, Wang CR, Yonekawa H, Hermel E, Lindahl KF. 1990. Maternally transmitted histocompatibilityantigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60: 971-80

Mannella CA. 2006a. The relevance of mitochondrial membrane topology to mitochondrial function. BiochimBiophys Acta 1762: 140-7

39

Mannella CA. 2006b. Structure and dynamics of thc mitochondrial inner membrane cristae. Biochim BiophysActa 1763: 542-8

McQuibban GA, Saurya S, Freeman M. 2003. Mitochondrial membrane remodelling regulatcd by aconserved rhomboid protease. Nature 423: 537-41

Meeusen SL, Nunnari J. 2005. How mitochondria fuse. Curr Opin Cell Biol 17: 389-94Mozdy AD, McCaffery JM, Shaw JM. 2000. Dnmlp GTPase-mediated mitochondrial fission is a multi-step

process requiring the novel intégral membrane component Fislp. J Cell Biol 151: 367-80Neuspiel M, Zunino R, Gangaraju S, Rippstein P, McBride HM. 2005. Activated Mfn2 signais mitochondrial

fusion, interfères with Bax activation and rcduces susceptibility to radical induced dcpolarization. JBiol Chem 280: 25060-70

Okamoto K, Shaw JM. 2005. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes.Annu Rev Genêt 39: 503-36

Olichon A, Emorine LJ, Descoins E, Pelloquin L, Brichese L, et al. 2002. The human dynamin-related proteinOPA1 is anchored to the mitochondrial inner membrane facing the inter-membranc space. FEBS Lett523: 171-6.

Pagliarini DJ, Wiley SE, Kimple ME, Dixon JR, Kelly P, et al. 2005. Involvement of a MitochondrialPhosphatase in the Régulation of ATP Production and Insulin Sécrétion in Pancreatic beta Cells. MolCell 19: 197-207

Pellegrini L, Passer BJ, Canelles M, Lefterov I, Ganjei JK, et al. 2001. PAMP and PARL, two novel putativemetalloproteases intcracting with the COOH-terminus of Presenilin-1 and -2. JAlzheimers Dis 3: 181-90

Rawson RB, Zelenski NG, Nijhawan D, Ye J, Sakai J, et al. 1997. Complémentation cloning of S2P, a gèneencoding a putative mctalloprotease required for intramembrane cleavage of SREBPs. Mol Ce/11: 47-57

Roche TE, Baker JC, Yan X, Hiromasa Y, Gong X, et al. 2001. Distinct rcgulatory properties of pyruvatedehydrogenase kinase and phosphatase isoforms. Prog Nucleic AcidRes Mol Biol 70: 33-75

Salvi M, Brunati AM, Toninello A. 2005. Tyrosine phosphorylation in mitochondria: a new frontier inmitochondrial signaling. Free Radie BiolMed3S: 1267-77

Schrader M. 2006. Shared components of mitochondrial and peroxisomal division. Biochim Biophys Acta1763: 531-41

Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, et al. 2002. A Distinct Pathway Remodcls MitochondrialCristae and Mobilizes Cytochrome c during Apoptosis. Dev Cell 2: 55-67

Sesaki H, Jensen RE. 2001. UGO1 encodes an outer membrane protein required for mitochondrial fusion. JCell Biol 152: 1123-34.

Sesaki H, Southard SM, Hobbs AE, Jensen RE. 2003. Cells lacking Pcplp/Ugo2p, a rhomboid-like proteaserequired for Mgmlp processing, lose mtDNA and mitochondrial structure in a Dnmlp-dependentmanner, but remain compétent for mitochondrial fusion. Biochem Biophys Res Commun 308: 276-83.

Shaw JM, Nunnari J. 2002. Mitochondrial dynamics and division in budding yeast. Trends Cell Biol 12: 178-84.

Shepard KA, Yaffe MP. 1999. The yeast dynamin-like protein, Mgmlp, functions on the mitochondrial outermembrane to médiate mitochondrial inheritance. J. Cell Biol. 144: 711-20

Shi Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9: 459-70Sik A, Passer BJ, Koonin EV, Pellegrini L. 2004. Self-regulated cleavage of the mitochondrial

intramembrane-cleaving protease PARL yields Pbeta, a nuclear-targeted peptide. J Biol Chem 279:15323-9.

Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. 1998. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization.MolCellX: 949-57

Steinmetz LM, Scharfe C, Deutschbauer AM, Mokranjac D, Herman ZS, et al. 2002. Systematic screen forhuman disease gènes in yeast. Nat Genêt 31: 400-4

Sugioka R, Shimizu S, Tsujimoto Y. 2004. Fzol, a protein involved in mitochondrial fusion, inhibits apoptosis.JBiol Chem 279: 52726-34. Epub 2004 Sep 30.

Traven A, Wong JM, Xu D, Sopta M, Ingles CJ. 2001. Interorganellar communication. Altered nuclear gèneexpression profiles in a yeast mitochondrial dna mut uni../ Mol Chem 276: 4020-7

Urban S. 2006. Rhomboid proteins: conserved membrane proteases with divergent biological functions. GènesDev 20: 3054-68

40

Urban S, Wolfe MS. 2005. Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid issufficient for catalysis and specificity. ProcNatlAcadSci USA 102: 1883-8

Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, et al. 2004. Hereditary early-onsetParkinson's discase caused by mutations in P1NK1. Science 304: 1158-60

Wang Y, Zhang Y, Ha Y. 2006. Crystal structure of a rhomboid family intramembrane protease. Nature 444:179-80

Weihofen A, Binns K, Lemberg MK, Ashman K, Martoglio B. 2002. Identification of signal peptide peptidase,a presenilin-type aspartic protease. Science 296: 2215-8

Weihofen A, Martoglio B. 2003. Intramembrane-cleaving protcases: controlled libération of proteins andbioactive peptides. Trends Cell Biol 13: 71-8

Wong ED, Wagner JA, Gorsich SW, McCaffery JM, Shaw JM, Nunnari J. 2000. The dynamin-relatedGTPase, Mgmlp, is an intermembrane space protein requircd for maintenance of fusion compétentmitochondria. J. Cell Biol. 151: 341-52

Wu Z, Yan N, Feng L, Oberstein A, Yan H, et al. 2006. Structural analysis of a rhomboid familyintramembrane protease reveals a gating mechanism for substrate entry. Nat Struct Mol Biol 13:1084-91

Yaffe MP. 2003. The cutting edge of mitochondrial fusion. Nat Cell Biol 5: 497-9Yoon Y, Krueger EW, Oswald BJ, McNiven MA. 2003. The mitochondrial protein hFisl régulâtes

mitochondrial fission in mammaiian cclls through an interaction with the dynamin-like proteinDLP1. Mol Cell Biol 23: 5409-20

Youle RJ, Karbowski M. 2005. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 6: 657-63Yu T, Fox RJ, Burwell LS, Yoon Y. 2005. Régulation of mitochondrial fission and apoptosis by the

mitochondrial outer membrane protein hFisl. J Cell Sci 118: 4141-51Zamzami N, Kroemer G. 2003. Apoptosis: mitochondrial membrane permeabilization—the (w)hole story?

Curr Biol 13: R71-3.Zhang J, Zheng N, Zhou P. 2003. Exploring the functional complexity of cellular proteins by protein

knockout. Proc Natl Acad Sci USA 100: 14127-32Zou H, Li Y, Liu X, Wang X. 1999. An APAF-l.cytochrome c multimeric complex is a functional apoptosome

that activâtes procaspase-9. J Biol Chem 274: 11549-56

41