Mechanisms of mitochondrial outer membrane permeabilization.

Abstract

In response to many apoptotic stimuli, Bcl-2 family pro-apoptotic members, such as Bax and Bak, are activated. This results in their oligomerization, permeabilization of the outer mitochondrial membrane, and release of many proteins that are normally confined in the mitochondrial inter-membrane space. Among these proteins are cytochrome c, Smac/DIABLO, OMI/HtrA2, AIF and endonuclease G. Mitochondrial outer membrane permeabilization (MOMP) is also associated with fragmentation of the mitochondrial network. The mechanisms that lead to the oligomerization of proapoptotic members of the Bcl-2 family and to MOMP are still unclear and the role of mitochondrial fission in these events remains elusive.

Full text

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MITOCHONDRIAL BIOLOGY:
NEW PERSPECTIVES

Page 3

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MITOCHONDRIAL
BIOLOGY: NEW
PERSPECTIVES
Novartis Foundation Symposium 287

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v
Contents
Symposium on New perspectives on mitochondrial biology, held at the Novartis Foundation,
London, 28–30 November 2006
Editors: Derek J. Chadwick (Organizer) and Jamie Goode
This symposium is based on a proposal by Michael Duchen
David G. Nicholls Chair’s introduction 1
Albert Neutzner, Richard J. Youle and Mariusz Karbowski
Outer mitochondrial membrane protein degradation by the proteasome 4
Discussion 14
Sarah E. Haigh, Gilad Twig, Anthony A. J. Molina, Jakob D. Wikstrom,
Motti Deutsch and Orian S. Shirihai PA-GFP: a window into
the subcellular adventures of the individual mitochondrion 21
Discussion 36
Luca Scorrano Multiple functions of mitochondria-shaping proteins 47
Discussion 55
Bruce M. Spiegelman Transcriptional control of mitochondrial energy
metabolism through the PGC1 coactivators 60
Discussion 63
Charles Affourtit, Paul G. Crichton, Nadeene Parker and Martin D. Brand
Novel uncoupling proteins 70
Discussion 80
Cecilia Giulivi Mitochondria as generators and targets of
nitric oxide 92
Discussion 100
György Hajnóczky, Masao Saotome, György Csordás, David Weaver and
Muqing Yi Calcium signalling and mitochondrial motility 105
Discussion 117

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vi CONTENTS
Anna Romagnoli, Paola Aguiari, Diego De Stefani, Sara Leo,
Saverio Marchi, Alessandro Rimessi, Erika Zecchini, Paolo Pinton
and Rosario Rizzuto Endoplasmic reticulum/mitochondria calcium
cross-talk 122
Discussion 131
Brian O’Rourke, Sonia Cortassa, Fadi Akar and Miguel Aon
Mitochondrial ion channels in cardiac function and dysfunction 140
Discussion 152
Paolo Bernardi and Michael Forte The mitochondrial permeability
transition pore 157
Discussion 164
Dominic James, Philippe A. Parone, Olivier Terradillos,
Safa Lucken-Ardjomande, Sylvie Montessuit and
Jean-Claude Martinou Mechanisms of mitochondrial outer membrane
permeabilization 170
Discussion 176
M. Flint Beal Mitochondria and neurodegeneration 183
Discussion 192
Mügen Terzioglu and Nils-Göran Larsson Mitochondrial dysfunction in
mammalian ageing 197
Discussion 208
Eric A. Schon and Salvatore DiMauro Mitochondrial mutations: genotype
to phenotype 214
Discussion 226
Contributor Index 234
Subject index 236

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vii
Participants
Vera Adam-Vizi Department of Medical Biochemistry, Semmelweis University,
PO Box 262, H-1444 Budapest, Hungary
M. Flint Beal Department of Neurology and Neuroscience, Weill Medical College
of Cornell University, 525 East 68th Street, Room F610, New York, NY 10021,
USA
Piotr Bednarczyk (Novartis Foundation Bursar) Department of Biophysics, Agri-
cultural University SGGW, 159 Nowousynowska St., 02-776 Warsaw, Poland
Paolo Bernardi Department of Biomedical Sciences, Università di Padova, Viale
Giuseppe Colombo 3, I-35121 Padova, Italy
Martin D. Brand MRC Dunn Human Nutrition Unit, Hills Road, Cambridge
CB2 2XY, UK
Michael Duchen Department of Physiology, University College London, Gower
Street, London, WC1E 6BT, UK
Cecilia Giulivi Department of Molecular Biosciences, University of California,
1311 Haring Hall, One Shields Avenue, Davis, CA 95616, USA
György Hajnóczky Department of Pathology, Anatomy and Cell Biology, Thomas
Jefferson University, Rm 253 7AH, 1020 Locust Street, Philadelphia, PA 19107,
USA
Andrew P. Halestrap Department of Biochemistry, School of Medical Sciences,
University of Bristol, Bristol BS8 1TD, UK
Derek Hausenloy (Novartis Foundation Bursar) The Hatter Cardiovascular Insti-
tute, University College Hospital, 67 Chenies Mews, London WC1E 6HX,
UK
Howard T. Jacobs Institute of Medical Technology, University of Tampere, FI
33014, Finland

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viii PARTICIPANTS
Nils-Göran Larsson Karolinska Institutet, Department of Laboratory Medicine,
Division of Metabolic Diseases, Novum, S-14186 Stockholm, Sweden
John J. Lemasters Pharmaceutical Sciences and Biochemistry & Molecular
Biology, Medical University of South Carolina, QF308 Quadrangle Building,
280 Calhoun Street, PO Box 250140, Charleston, SC 29425, USA
Jean-Claude Martinou Department of Cell Biology, Sciences III, University of
Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland
David G. Nicholls (Chair) Morphology Core, Buck Institute for Age Research,
8001 Redwood Blvd, Novato, CA 94945, USA
Brian O’Rourke School of Medicine, Institute of Molecular Cardiobiology, The
Johns Hopkins University, 720 Rutland Avenue, 1059 Ross Bldg., Baltimore,
MD 21205-2195, USA
Sten Orrenius Institute of Environmental Medicine, Division of Toxicology,
Karolinska Institutet, Box 210, Stockholm, SE-171 77, Sweden
Anant Parekh Department of Physiology, Anatomy and Genetics, University of
Oxford, Parks Road, Oxford, OX1 3PY, UK
Ian J. Reynolds Merck Research Laboratories, WP42-229, 770 Sumneytown Pike,
P O Box 4, West Point, PA 19486-0004, USA
Peter R. Rich The Glynn Laboratory of Bioenergetics, Department of Biology,
University College London, Gower Street, London WC1E 6BT, UK
Rosario Rizzuto Department of Experimental and Diagnostic Medicine, General
Pathology Section, University of Ferrara, Via L. Borsari 46, 44100 Ferrara,
Italy
Eric A. Schon Department of Neurology, Room 4-431, College of Physicians and
Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032,
USA
Luca Scorrano Dulbecco-Telethon Institute, Venetian Institute of Molecular
Medicine, Via Orus 2, Padova, I-35129, Italy

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PARTICIPANTS ix
Orian Shirihai Department of Pharmacology and Experimental Therapeutics,
Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111,
USA
Bruce M. Spiegelman Dana-Farber Cancer Institute and Department of Cell
Biology, Harvard Medical School, Boston, MA 02115, USA
Douglas M. Turnbull Mitochondrial Research Group, School of Neurology,
Neurobiology and Psychiatry, The Medical School, Newcastle University,
Newcastle Upon Tyne, NE2 4HH, UK
Richard J. Youle Biochemistry Section, Surgical Neurology Branch, National
Institutes of Health, Bldg 35, Room 2C917, 25 Convent Drive, MSC 370,
Bethesda, MD 20892, USA

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Page 12

1
Chair’s introduction
David G. Nicholls
Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945, USA
In this introduction I want to summarize where we are in the fi eld and where we
are going. What is the future of mitochondrial bioenergetics? A couple of weeks
ago I had an idle moment, so I logged on to PubMed and entered the search term
‘mitochondria’ followed by the years 1950 and 2006, one after the other. The
results were fascinating. The numbers of citations per year for mitochondria started
off in the bioenergetic prehistory, going back almost 100 years to the fi rst descrip-
tions of mitochondria. For me, the single event that introduced the classic era of
mitochondrial bioenergetics was the publication of papers by Chance and Williams
in the mid 1950s which fi rst described the oxygen electrode, and described the
redox changes of the cytochromes. What happened then was an explosive growth
over the next 10 or 15 years in the number of papers, which led to over 3000 papers
per year by the time the next revolution came. This was Peter Mitchell’s work in
the late 1960s. This is interesting: whereas you would expect that the discovery of
a mechanism would stimulate a lot of new research, what happened after the three
or four years when Peter was publishing these fantastic papers is that the fi eld
stagnated for 20 years in terms of numbers of publications. Somehow, because
bioenergetics had defi ned itself so narrowly in terms of understanding how the
respiratory chain and ATP synthase works with a little bit of ion transport, this
limited the fi eld.
The next explosion of research came when our cell biology colleagues working
on cell health and death discovered, sometimes to their discomfort, that mitochon-
dria moved into the centre of the fi eld. In the last 10 years the trend has been
almost explosive in terms of the number of papers on mitochondrial bioenergetics.
I didn’t have time to do a statistical sampling of the different years, but my guess
is that 80% of these papers come under the fi eld of mitochondrial physiology:
mitochondria in the context of the cell.
What is interesting is where we are going. To quote Donald Rumsfeld, it is the
unknown unknowns that will defi ne where the fi eld moves in the next 10 years.
In 1994, no one knew that they didn’t know how cytochrome c was released,
because it wasn’t part of the vocabulary. What does this have to do with cell death?
It is the things that we don’t know that we don’t know which are going to defi ne
the next 10 years or so.

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2 NICHOLLS
What Michael Duchen has organized for this meeting is a series of four separate
sessions. The fi rst one is mitochondrial ‘natural history’, a phrase I like because
it conjures up images of David Attenborough wandering through the cell
and getting excited when he comes close to a mitochondrion. The three papers
in this session deal with mitochondrial morphology, fi ssion, fusion, replacement
of proteins and shape. Some of the questions that occurred to me and which we
will be dealing with are as follows. Do you repair a mitochondrion, or are they
scrapped and replaced when they go wrong? How is a damaged mitochondrion
recognized, and why doesn’t this recognition work well in terms of mitochondrial
genetic disease? Do we see more fi ssion than fusion if there are more mitochondria
being produced, and what is the relationship between fi ssion/fusion and
biogenesis?
The second session is mitochondria and oxidative stress. I’m fascinated by
PGC1α, and equally fascinated by novel uncoupling proteins. Does PGC1α regu-
late gross mitochondrial bioenergetics? Does it regulate the specifi c induction of
antioxidant pathways? What are the novel uncoupling proteins really doing? This
is still surprisingly cloudy, some 10 years after the fi rst description of these
proteins.
The third session deals with signal transduction. Mitochondrial nitric oxide
synthase is still controversial. Mitochondrial Ca2+ signalling and endoplasmic
reticulum (ER) cross-talk is also a hot topic: what are the conditions where the
ER and mitochondria talk to each other, and what are the conditions where they
work on their own? Ion channels will be covered: one ion channel that is highly
controversial is the mitochondrial K+-ATP channel. What is the problem with
this? Is it present or not? The permeability transition pore is another subject for
discussion: when is it important and when is it not? What are the proven cases for
its involvement and what are the more speculative cases? With regard to the regula-
tion of cytochrome c release, do we have to look at this separately from the release
of AIF and other proteins? Is there a single, holistic mechanism for releasing these
proapoptotic proteins from mitochondria or is cytochrome c a special case? Mito-
chondria and neurodegeneration is an enormous topic: one of the central questions
here is why does damage in these different diseases show such tissue specifi city?
Is it related to the cell or the mitochondrion itself?
The fi nal session looks at mitochondrial mutations. Even with the Pol-g muta-
tions, is the frequency of mitochondrial DNA mutations suffi cient to account for
the phenotype? That is, is there any disconnect between the proportion of mito-
chondria that possess the mutation and the dysfunctional phenotype? Why do the
mechanisms of autophagocytosis not recognize some damaged mitochondria when
there is heteroplasmic coexistence of normal and damaged mitochondria? This
seems to work in the β cell; what goes wrong in some mitochondrial diseases where
it goes the wrong way, and the mutated mitochondria seem to be dominant over

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CHAIR’S INTRODUCTION 3
the wild-type ones? Also, why do we start off each generation with perfect mito-
chondria? What are the mechanisms that protect the germ cell or allow a Darwin-
ian-style selection of perfect mitochondria in each succeeding generation? Finally,
where will the next 10 years take us? We clearly don’t know the unknown unknowns,
but thinking of the known unknowns do we have a feel for where we are going?
I feel that part of the future lies in understanding mitochondrial morphology: why
are they shaped the way they are, and why do we have exactly the right number in
a cell? Why do they appear to go where they are needed?

Page 15

4
Outer mitochondrial membrane protein
degradation by the proteasome
Albert Neutzner*, Richard J. Youle* and Mariusz Karbowski*†
*Biochemistry Section, SNB, NINDS, NIH Bethesda MD 20892 and †Medical Biotechnology Center,
University of Maryland Biotechnology Institute, University of Maryland, 725 W. Lombard St, Baltimore,
MD 21201, USA
Abstract. Protein turnover is used for regulatory processes and to eliminate superfl uous,
denatured or chemically inactivated polypeptides. Mitochondrial proteins may be par-
ticularly susceptible to damage induced by reactive oxygen species and several pathways
of mitochondrial proteolysis have been illuminated. However, in contrast to matrix
and inner mitochondrial membrane protein degradation, little is known about the
turnover of integral outer mitochondrial membrane (OMM) proteins or the mechanisms
involved. We have found that pheromone treatment of Saccharomyces cerevisiae induces the
proteasome-dependent elimination of the OMM spanning protein, Fzo1, from the
mitochondria and that Fzo1 is ubiquitylated while still associated with the membrane.
These characteristic processing steps are similar to those of the endoplasmic reticulum
(ER)-associated degradation (ERAD) pathway suggesting the term OMMAD, outer
mitochondrial membrane-associated degradation, to describe the process. ERAD is
dependent upon ER membrane spanning RING domain E3 ubiquitin ligases suggesting
that certain E3 ligases in the OMM may also regulate OMMAD. This led us to clone
and characterize all 54 predicted human gene products that contain both RING domains
and predicted membrane spanning domains. A surprising number of these localize to
mitochondria where some may control OMMAD. Some of these mitochondrial RING
domain proteins also regulate mitochondrial morphology, indicating a critical role of
ubiquitin signalling in the maintenance of mitochondrial homeostasis.
2007 Mitochondrial biology: new perspectives. Wiley, Chichester (Novartis Foundation Symposium
287) p 4–20
Regulated protein degradation is used to eliminate superfl uous, denatured or
chemically inactivated polypeptides and in signal transduction. Current evidence
shows that in the mitochondria, protein degradation is controlled by pathways
compartmentalized in the matrix and the inner mitochondrial membrane. For
example, it has been demonstrated that Pim1/Lon proteases, of the AAA protease
family homologous to bacterial proteases, degrade excess soluble matrix proteins
whereas membrane embedded i- and m-AAA proteases remove integral membrane
proteins in the inner mitochondrial membrane. Additional components of this