fission processes

The International Journal of Biochemistry & Cell Biology 41 (2009) 1828–1836

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Review

Importance of lipid metabolism for intracellular and mitochondrial membrane fusion/fission processes Fabienne Furt, Patrick Moreau ∗ Membrane Biogenesis Laboratory, UMR 5200, University of Bordeaux II-CNRS, France

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Article history: Available online 20 February 2009 Keywords: Mitochondria dynamics Membrane fusion Membrane fission Lipid metabolism

a b s t r a c t Mitochondria move along cytoskeletal tracks, fuse and divide. These dynamic features have been shown to be critical for several mitochondrial functions in cell viability and cell death. After a rapid recall of the proteic machineries that are known to be involved, the review will focus on lipids, other key molecular actors of membrane dynamics. A summary of the current knowledge on lipids and their implication in various cellular membrane fusion/fission processes will be first presented. The review will then report what has been discovered or can be expected on the role of the different families of lipids in mitochondrial membrane fusion and fission processes. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fusion and fission of mitochondrial membranes: the protein machineries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mitochondrial fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mitochondrial fission proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Regulation of mitochondrial fusion/fission proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fusion and fission of mitochondrial membranes: lipid-assisted processes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. From the proteic to the lipidic world . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Lipid-assisted intracellular membrane dynamics and trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Physicochemical properties of lipids in membrane dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Lipid-assisted mitochondrial membrane dynamics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. CL and the other phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Neutral lipids and sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mitochondria are complex intracellular organelles of prokaryotic origin, which are required for numerous critical cell metabolic functions. Mitochondria for example produce energy by oxidative phosphorylation, are involved in iron and calcium homeostasis, in

Abbreviations: CL, cardiolipin; DAG, diacylglycerol; LPA, lyso-phosphatidic acid; LPC, lyso-phosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; SM, sphingomyelin. ∗ Corresponding author at: Laboratoire de Biogenèse Membranaire, UMR CNRS 5200, Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat, Case 92, 33076 Bordeaux Cedex, France. Tel.: +33 5 57 57 16 81; fax: +33 5 56 51 83 61. E-mail address: [email protected] (P. Moreau). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.02.005

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amino acid, fatty acid and steroid hormone metabolism, and are also actors of cell apoptosis (Meeusen and Nunnari, 2005; Westermann, 2008). Mitochondria are dynamic structures that show different morphologies: small spheres, short rods or long tubules which depend on cell type and also cell status (Detmer and Chan, 2007; Benard and Rossignol, 2008; Westermann, 2008). In most eukaryotic cells, mitochondria move along cytoskeletal tracks and their overall morphology depend on the balance between fusion and fission events. For example, an extent of fusion activities leads to interconnected mitochondrial networks, and on the contrary, an extent of fission events generates numerous different small spherical organelles. Some aspects of the morphology of the mitochondrial network in animal cells are shown in Fig. 1 (see also Benard and Rossignol, 2008). In both cases, the organisation and dynamics of mitochondria are related to critical physiological functions

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Fig. 1. Morphology of the mitochondrial network in animal cells. Among the diversity of the morphologies of the mitochondrial network in Hela cells (Benard and Rossignol, 2008), four frequent shapes are presented: the classic reticular network, the intermediate, the fragmented, and the outgrowth.

(Benard and Rossignol, 2008; Westermann, 2008). For example, an extended tubular mitochondrial network can be engaged in calcium signalling, energy dissipation, apoptosis and defense against aging (still hypothetical), whereas disassembled mitochondria are required for cytokinesis or apoptosis (Meeusen and Nunnari, 2005; Benard and Rossignol, 2008; Westermann, 2008). Perturbations of mitochondrial dynamics can have tremendous consequences on cell metabolism and therefore on cell life/cell death (Meeusen and Nunnari, 2005; Jensen and Sesaki, 2006; Detmer and Chan, 2007; Benard and Rossignol, 2008; Benard et al., 2008; Parone et al., 2008 and references therein). It has been described that mice defective in mitochondrial fusion cannot sustain development and die, yeast mutants also defective in mitochondrial fusion lose their mitochondrial DNA and cannot run oxidative phosphorylations, similar perturbations of human cell mitochondria dynamics (fusion or fission) lead to numerous disorders (Charcot-Marie-Tooth 2A or 4A, autosomal dominant optic atrophy or neonatal lethality). In addition, the frequent fusion/fission events undergone by the mitochondrial network appears clearly linked to the bioenergetic state of mitochondria (Benard and Rossignol, 2008; Benard et al., 2008; Twig et al., 2008). The compartmentalization of the substrates of the respiratory chain, the physical seggregation of mitochondrial domains in membrane tubules, and a control of mitochondria homeostasis by autophagy (Benard and Rossignol, 2008; Benard et al., 2008; Twig et al., 2008) certainly involve mitochondria dynamics and related fusion/fission events. As a consequence, unravelling the mechanisms and the machineries conducting mitochondrial fusion and fission processes which govern the balance between the different mitochondrial morphologies, is critical for our understanding of mitochondrial dynamics and their impact on cell physiological functions. After a rapid summary of the proteic machineries that have been demonstrated to be involved in mitochondria dynamics, the review will focus on lipids, the other key molecular actors of membrane dynamic processes. A first part will present our current knowledge on lipids and their implication in various cellular membrane fusion/fission processes. We will then report what has been discov-

ered or can be expected on the role of the different families of lipids in mitochondrial membrane fusion and fission processes. 2. Fusion and fission of mitochondrial membranes: the protein machineries Genetic and biochemical studies in several organisms (drosophila, yeast and mammalian cells) have contributed to identify and characterize the major components of the fusion and fission machineries involved in mitochondria dynamics and their regulation (Meeusen and Nunnari, 2004, 2005; Malka et al., 2005; Nakamura et al., 2006; Boldogh and Pon, 2007; Cerveni et al., 2007; Coonrod et al., 2007; Detmer and Chan, 2007; Song et al., 2007; Martens and McMahon, 2008; Merkwirth et al., 2008; Poole et al., 2008; Westermann, 2008; Wickner and Schekman, 2008 and references therein). A summary of the main proteins discovered and characterized in yeast and mammals is given in Table 1. 2.1. Mitochondrial fusion proteins Membrane fusion events require first that the maintainedintermembrane distance due to electrostatic repulsion be reduced. Specific proteins will pull the opposed membrane bilayers together to induce bent membranes and/or protein-depleted areas where fusion can proceed (Lang et al., 2008). Then, these or other proteins will stabilize the stalk intermediates and generate fusion pores (Lang et al., 2008). One viral fusion protein can generally assume all of these steps. On the contrary, intracellular fusion events (including mitochondrial fusion) need several protein complexes to control the successive steps. Among intracellular membrane fusion processes, mitochondrial fusion is unusual insofar as it requires the successive fusions of outer and inner membranes, and is therefore different from the events that govern membrane fusion in the secretory pathway for example. In addition, classical fusion proteins in the secretory pathway such as SNAREs (proteins involved in membrane fusion through the secretory pathway) are not involved, and specific proteins have emerged in the mitochondrial context.

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Table 1 Major proteins required for mitochondrial fusion and fission mechanisms. Proteins (Yeast-mammals)

Protein family

Localisation

Functions

Fzo1-Mitofusins 1/2 Mgm1-Opa1 Ugo1-? Dnm1-Drp1 Fis1-hFis1 Caf4/Mdv1-? Mdm33-Mtp18

Dynamin-related GTPases Dynamin-related GTPases MCP family Dynamin-related GTPases Membrane receptor WD40 repeat region containing proteins Coiled-coil rich proteins

OM IM–IMS OM Cytosol OM Cytosol IM

Mitochondria tethering Outer membrane fusion Inner membrane fusion Links Fzo1 to Mgm1 to coordinate OM-IM fusions OM scission Dnm1/Drp1 membrane anchor Fis1 Dnm1 adaptors IM scission

IM: inner membrane; IMS: inter-membrane space; MCP: mitochondrial carrier proteins; OM: outer membrane.

The yeast Fzo1, and the mammal mitofusins 1 and 2 which are dynamin-related GTPases of the outer membrane, are engaged in mitochondria tethering and outer membrane fusion. The tethering function of mitofusins is mediated by their C-terminal ␣-helices which can form antiparallel coiled-coils. The subsequent fusion activity of mitofusins is driven by their N-terminal GTPase domains. It is believed that the ability of these proteins to favor membrane tubulation may be required as a driving force for conducting fusion, but insofar as the extension of tubule formation is controlled in a coordinated fashion by the proteic machinery. Mgm1 and Ugo1 in yeast and the Mgm1 ortholog Opa1 in mammals are believed to coordinate the outer membrane fusion step with the inner membrane contacts thought to be necessary for the inner membrane fusion process itself. Mgm1 and its ortholog Opa1 are dynamin-like GTPases that can have a direct physical involvement in the fusion of inner membranes. According to complementation studies and in vitro fusion assays developed recently, it has been postulated that Mgm1/Opa1 may oligomerize to drive a SNARE-like governed interaction of the inner membranes to be fused. On another hand it can also be proposed that these dynamin-related GTPases function through formation of tubule extensions with curved ends as mentioned for mitofusins and endocytic dynamin. Ugo1 is found in the outer membrane and is proposed to link Fzo1 of the outer membrane with Mgm1 of the inner membrane in yeast, and due to its multipass topology may assemble into a fusion pore (Coonrod et al., 2007). Fzo1 was found in contact sites between outer and inner membranes, and in protein complexes with Ugo1 and Mgm1. Therefore, these features argue in favor of an expected coordinated action of the successive fusion machineries to ensure double membrane fusion. However, no homolog of Ugo1 has yet been identified in mammals to have a similar function with mitofusins and Opa1. As a consequence, the concept of a coordinated fusion between outer and inner membranes in mammals was questioned, and indeed a specific assay developed in living human cells argued that outer membrane and inner membrane fusion machineries can proceed separately (Malka et al., 2005). For example, inner membrane fusion was hampered by dissipating membrane potential with K+ and H+ ionophores or inhibition of glycolysis whereas the fusion of outer membranes was insensitive or less sensitive (Malka et al., 2005). In addition, it has been shown in mouse embryonic fibroblasts that mitochondrial fusion is regulated by the formation of long and short proteolytic-processed isoforms of Opa1, and that stability of long isoforms are sensitive to membrane potential (Song et al., 2007). It is interesting to note that outer membrane fusion requires the proton gradient component of the inner membrane electrochemical potential but not its electrical part, however the electrical component is critical for inner membrane fusion (Meeusen and Nunnari, 2004, 2005). A recently proposed model has established a critical flux–force relationship between oxidative phosphorylations/bioenergetic status of mitochondria and their network dynamic state (Benard et al., 2007), but such a link may not concern inner and outer membranes to the same extent. We can consider that proteins have still to be discovered in the mammal machineries and/or that substantial differences exist between

yeast and mammal mechanisms of mitochondrial dynamics behind common principles. 2.2. Mitochondrial fission proteins Four major proteins contribute to mitochondrial membrane fission in yeast: the outer membrane protein Fis1 and the three cytosolic proteins Caf4, Dnm1 and Mdv1 (Westermann, 2008). Fis1 and its human ortholog named hfis1 are tailed-anchored proteins considered as receptors for the recruitment of cytosolic components of the outer fission machinery. Dnm1 and its mammal ortholog DRP1/DLP1 are dynamin-like proteins containing an Nterminal GTPase domain and a C-terminal GTPase effector domain. Caf4 and Mdv1 are related proteins with redundant functions. They have two N-terminal ␣-helices which interact with Fis1, a C-terminal WD40 repeat domain that may bind Dnm1 and a coiledcoil region that may participate to self-oligomeric interactions. No orthologs of these two proteins have yet been identified in mammals. Dnm1 seems to be the scission protein of the outer mitochondrial membrane (recruited by Fis1 and Caf4/Mdv1), and its GTP hydrolysis-dependent activity in driving membrane fission looks similar to what is known for dynamin-dependent membrane fission in endocytosis for example. Considerations on the mean diameter of mitochondrial tubules (300–400 nm) and Dnm1 spirals (about 100 nm), and the characterization of dnm1 deletion mutants and drp1 mutants in C. elegans point to the requirement of inner membrane fission to produce constriction events indispensable for outer membrane fission to proceed (Westermann, 2008). The inner membrane proteins Mdm33 in yeast and Mtp18 in mammals are believed to favor inner membrane fission because overexpression or depletion of these proteins induces either a fragmentation of the mitochondrial network or the accumulation of giant mitochondria. However, since no functional assay is available in vitro or in vivo to assess the role of these proteins, it is not known whether their participation is direct or indirect. As compared to mitochondrial membrane fusion, more has to be learned for clarifying the mechanisms of mitochondrial membrane fission. 2.3. Regulation of mitochondrial fusion/fission proteins Numerous proteins and regulatory mechanisms have been evidenced to be involved in the regulation of fusion/fission proteins (Meeusen and Nunnari, 2004, 2005; Nakamura et al., 2006; Boldogh and Pon, 2007; Cerveni et al., 2007; Merkwirth et al., 2008; Poole et al., 2008; Westermann, 2008 and references therein). For a list of the various proteins discovered, the readers may consult the Table 1 in Cerveni et al. (2007). Some important features are for example the control of mitochondrial morphology through the regulation by MARCH-V of both mitofusins and the ubiquitinated forms of Drp1 (Nakamura et al., 2006), or the regulation of the assembly of mitofusin complexes and the recruitment of Drp1 to the outer membrane by the pro-apoptotic proteins Bax ad Bak and novel mitofusin-binding proteins (Cerveni et al., 2007). Insights into the diverse modes of regulation of the fusion–fission balance of mito-

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chondrial dynamics will help to unravel the mechanisms which link variations of mitochondrial morphology with cellular processes such as cell division, cell growth, apoptosis and their connection with human diseases. 3. Fusion and fission of mitochondrial membranes: lipid-assisted processes? 3.1. From the proteic to the lipidic world Beside the protein machineries, more and more data implicate lipids, enzymes of lipid metabolism and lipid modifications of proteins in the mechanisms of membrane dynamics and their regulation. Lipids have been shown to act as modulators of viral protein-mediated membrane fusion. For example, membrane fusion required for virus entry can be dependent on cholesterol, sphingolipids and lipid microdomains (Teissier and Pécheur, 2007). Mitochondrial membrane fusion is also concerned by these features since yeast screens identified all the enzymes of the ergosterol biosynthetic pathway (coded by Erg genes) as critical factors involved in mitochondrial morphology (Dimmer et al., 2002; Altmann and Westermann, 2005). Since the concentration of ergosterol is very low in mitochondrial membranes (6–25 ␮g/mg proteins), it was proposed that a regulation of the fusion/fission proteic machineries by ergosterol rather than a direct modulation of the physical properties of membranes was the impact of ergosterol biosynthesis on mitochondrial morphogenesis (Altmann and Westermann, 2005). However, it must be considered that variations of less than 1% in the amounts of a given lipid, if concentrated in a specific domain, may have critical impact on the local membrane structure. More recently a specific mitochondrial phospholipase D in the outer membrane has also been found to be a key actor of mitochondrial fusion by generating PA from CL (Choi et al., 2006). These data indicate some similarities between lipid requirement in mitochondrial fusion and SNARE-mediated fusion of other intracellular membranes. In addition, the fatty acyl transferase endophilin B1 was reported to be involved in the maintenance of mitochondrial morphology (Karbowski et al., 2004). Therefore lipids are also very critical actors of membrane dynamics. In the following sections we will first describe our current knowledge on the role of lipids in general intracellular membrane dynamics and trafficking, present the physicochemical bases of lipid properties for membrane dynamics, and then estimate what the different lipids can do in mitochondrial fusion/fission events. 3.2. Lipid-assisted intracellular membrane dynamics and trafficking Cholesterol has been shown to be critical in the formation of post-Golgi secretory vesicles (Wang et al., 2000), and may also be required to help vesicle fusion (Churchward et al., 2005). In addition, it has been found that cholesterol levels in the Golgi membranes must be tightly regulated since an excess of this molecule can lead to a vesiculation of the Golgi complex itself (Grimmer et al., 2005). Another example in yeast clearly established that ergosterol is critical for endocytosis (Heese-Peck et al., 2002). In plants, blocking phytosterol maturation in the ER by the drug fenpropimorph (a plant specific inhibitor of the cycloeucalenol-obtusifoliol isomerase) induced a fenestration of the Golgi bodies (Hartmann et al., 2002). On another hand, affecting the morphology of these Golgi bodies by brefeldin A treatment, which disturbs the recruitment of COPI proteins to the Golgi, reduced the biosynthesis of phytosterols (Mérigout et al., 2002). Therefore a strong relationship does exist between sterol metabolism and Golgi morphology in plant cells and certainly in most eukaryotic cells. As a consequence, and similarly

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as ergosterol in yeast, we could speculate about the critical role that cholesterol and phytosterols may have on animal and plant mitochondrial morphology. The sphingolipid family may also affect the organisation of endomembranes. In animal cells, ceramides dramatically increase the disassembly of the Golgi that is induced by brefeldin A, which suggests that changes in ceramide levels affect the stability of the Golgi (Fukunaga et al., 2000). Pharmacological approaches using inhibitors of glucosylceramide synthase argue in favor of a role of sphingolipids in the architecture of the Golgi complex (Nakamura et al., 2001). In addition, high concentrations of the long chain base (LCB) sphingosine derived from ceramide hydrolysis can induce the fragmentation of the Golgi complex (Hu et al., 2005). In yeast, C26sphingolipids were found to be required for correct transport of the Pma1 ATPase to the plasma membrane (Gaigg et al., 2005). In plant cells, fenpropimorph treatment led to the accumulation of both phytosterol precursors and hydroxypalmitic acid-containing glucosylceramide in the Golgi membranes (Laloi et al., 2007). Therefore, as sterols, sphingolipids may be critical for Golgi morphodynamics and their implication in the function of the secretory pathway. As a consequence, since sterols and sphingolipids can be associated to form lipid microdomains in eukaryotic cells (Bagnat and Simons, 2002; Simons and Vaz, 2004; Laloi et al., 2007 and references therein), these features point to the importance of lipid domain formation in the efficiency of the secretory pathway (Bagnat and Simons, 2002; Simons and Vaz, 2004; Hancock, 2006; Laloi et al., 2007 and references therein; and see below). Other lipid families are also regulating endomembrane morphodynamics. Lysophospholipids, formed by phospholipases A2, can induce membrane tubulation (De Figueiredo et al., 1998), and inhibition of the reverse reaction by acyltransferases also leads to Golgi membrane tubulation (Drecktrah et al., 2003). Inhibition of such phospholipases A2 by specific inhibitors can for example block endosome fusion (Mayorga et al., 1993). Phospholipases A1related enzymes have been shown to induce a dispersion of the Golgi complex and aggregation of ER membranes (Nakajima et al., 2002). Inhibition of phospholipase D, which decreases PA, can also affect the structural integrity of the Golgi complex in animal cells (Siddhanta et al., 2000). Recently, it has been demonstrated that PA produced by the phospholipase isoform D2 is critical for the release of Golgi-derived COPI vesicles (Yang et al., 2008). The phospholipase D2 isoform is indeed concentrated to the rims of the Golgi stacks and could be involved in the Golgi morphodynamics that may drive some aspects of post-Golgi trafficking (Freyberg et al., 2002) and the stability of Golgi structure (Yang et al., 2008). Together with PA, PIP2 is another critical acidic phospholipid for membrane fusion (Vicogne et al., 2006 and references therein). Moreover, it was observed from in vitro SNARE-reconstituted fusion assays in liposomes that the highest fusion efficiency was obtained when PA was present in the acceptor liposomes (containing the tSNAREs syntaxin 4 and SNAP33) and PIP2 was included in the donor liposomes (containing the v-SNARE VAMP2) (Vicogne et al., 2006). These in vitro assays indicate that an asymmetric distribution of acidic phospholipids between the membranes to be fused may participate in vivo to the regulation of membrane fusion (Vicogne et al., 2006). In plant cells, inhibition of phospholipase D and therefore formation of PA in the pollen tube highly reduces the number of secretory vesicles, suggesting that PA levels may regulate pollen tube growth (Potocky et al., 2003; Monteiro et al., 2005). In addition, we have observed that the inhibition of phytosterol maturation by fenpropimorph was accompanied by an increase of PA in the endomembranes and particularly the Golgi (Moreau, 2007), which may suggest that elevated PA levels were at the origin of the fenestration of the Golgi bodies (Hartmann et al., 2002). In animal cells, it has recently been shown that the phospholipid-binding protein Nir2 (a Sec14-like protein) is required in the control of DAG lev-

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els in mammalian Golgi membranes (Litvak et al., 2005) as it was earlier proposed in yeast (Kearns et al., 1997), and this could be related to the formation of post-Golgi secretory vesicles. In addition, it has also been suggested that DAG may be required in the formation and fission of Golgi-derived COPI vesicles involved in Golgi-ER retrograde transport, through the recruitment of the protein ARFGAP1 (Fernandez-Ulibarri et al., 2007) and its own physicochemical properties. Protein sorting towards the secretory pathway in eukaryotic cells may be assisted by lipid-based sorting events and also specific interactions with acidic phospholipids (Ceppi et al., 2005; Lev, 2006). For example, a specific targeting of PS to ER-derived domains was evidenced in plant cells (Vincent et al., 2001), and PS was demonstrated to be required in the formation of ER-derived COPII vesicles in vitro (Matsuoka et al., 1998). Short chain ceramides can influence the binding of ARF to Golgi membranes and therefore affect the formation of COPI vesicles (Abousalham et al., 2002). In yeast, the oxysterol-binding protein (OSBP) Kes1p is a lipid receptor which may regulate the formation of Golgi secretory vesicles through regulation of the ARF and Sec14 pathways (Li et al., 2002). In animal cells, OSBP-related proteins have been shown to interact with a syntaxin-like VAMP-associated protein-A, and to be involved in the function of the COPII-dependent ER-Golgi anterograde transport pathway required in protein and ceramide transport (Wyles et al., 2002). Phospholipase D and phospholipase A1-like enzymes can interact with proteins of the COPII machinery to sustain the formation and regulation of ER export sites (Pathre et al., 2003; Shimoi et al., 2005). It has recently been observed that SNAREs can have different affinities for specific lipid microdomains such as sterol- and sphingolipid-rich lipid rafts, and that the degree of their association with these domains may regulate the efficiency of exocytosis (Salaün et al., 2005a,b). Moreover the basic domain of the synaptobrevin VAMP2 strongly interacts with the phospholipid PS (De Haro et al., 2003). Acidic phospholipids (PA, PIP2, PS) may regulate the formation of SNARE complexes and therefore the fusion events required for secretion to occur. Finally, it has recently been proposed that ceramides produced from sphingolipid-rich domains contribute to the lateral segregation and sorting of protein cargos within endosomal membranes (Trajkovic et al., 2008). The interactions between lipids and proteins of the intracellular transport machineries may therefore contribute to regulate the specific targeting and function of these proteins. It can be considered that at the time a lipid is formed, its physicochemical properties will spontaneously allow or avoid specific interactions with other partners (lipids or proteins) that will influence its association within a microdomain or another, and according to this environment, will target the lipid and its partners to a specific transport pathway. Therefore lipids and lipid-modifying enzymes must be considered as key regulators of membrane domain formation and homeostasis which strongly regulate membrane morphodynamics and trafficking. A brief summary of the relationships between endomembrane morphodynamics and lipid metabolism is given in the Table 2. With protein-based machineries, lipid-based machineries are therefore crucial regulatory forces also related to protein trafficking and sorting through the secretory pathway. We will now address some biophysical aspects of membrane organisation and deformation to rely these events with the physicochemical properties of lipids. 3.3. Physicochemical properties of lipids in membrane dynamics Although fusion and fission proteic machineries of intracellular and mitochondrial membranes are different, fission can be considered, at least in part, as two simultaneous fusion events. Therefore, on the lipid side we will consider that the properties of lipids involved in membrane fusion/fission processes are similar, and dis-

Table 2 Lipids and lipid metabolism involved in intracellular membrane morphodynamics. Lipids

Metabolism

Membrane dynamics

Cer

[Cer] high Cer formation by SMase

Golgi vesiculation Budding of exosome vesicles

DAG

[DAG] high [DAG] low

Golgi vesiculation Inhibition of Golgi budding

FFA LCB

FFA formation by PLA2 inhibited [LCB] high

Inhibition of endosome fusion Golgi vesiculation

LysoPL

[LPC] high LPA acylation to PA inhibited

ER/Golgi tubulation Inhibition of Golgi budding

PA

[PA] high [PA] low [PA] increase in AL [PA] increase in DL

Golgi vesiculation, fenestration Inhibition of Golgi budding Stimulation of DL/AL fusion Inhibition of DL/AL fusion

PIP2

[PIP2] increase in DL [PIP2] increase in AL

Stimulation of DL/AL fusion Inhibition of DL/AL fusion

Sterols

[Sterols] high [Sterols] low

Golgi vesiculation Golgi fenestration Inhibition of Golgi budding

AL: acceptor liposomes in SNARE-reconstituted in vitro fusion assay; Cer: ceramides; DAG: diacylglycerol; DL: donor liposomes in SNARE-reconstituted in vitro fusion assay; ER: endoplasmic reticulum; FFA: free fatty acids; LCB: long chain bases; LPA: lysophosphatidic acid; LPC: lysophosphatidylcholine; LysoPL: lysophospholipids; PLA2: phospholipase A2 which hydrolyzes major phospholipids at the sn2 position; PM: plasma membrane; SMase: sphyngomyelinase; SV: secretory vesicles.

cuss the roles of lipids in membrane fusion. Different putative roles can be attributed to lipids in membrane fusion processes (Lang et al., 2008). Lipids can serve as protein receptors through ionic interactions and therefore contribute to the recruitment of cytosolic proteins of the machineries, they can modify proteins for their targeting and/or function (see also Haucke and Di Paolo, 2007), they can help the lateral seggregation of proteic partners into specific domains, and they can contribute to the biophysical properties of the membranes to be fused at several levels (i.e. membrane curvature, non lamellar intermediary phases . . ., see below). In addition, lipid-induced membrane curvature can also condition the recruitment and activity of proteic partners (Bigay et al., 2005; Mesmin et al., 2007), and protein–lipid interactions can, as in the case of syntaxin1A, contributes to concentrate fusogenic lipids such as PA to the fusion sites (Lam et al., 2008). Several recent reviews have detailed the biophysical properties of the protein- and lipid-based machineries that sustain the mechanisms of membrane fusion (Janmey and Kinnunen, 2006; Haucke and Di Paolo, 2007; Chernomordik and Kozlov, 2008; Lang et al., 2008; Martens and McMahon, 2008; Wickner and Schekman, 2008). It is now mostly accepted that membrane fusion proceeds through a hemifusion intermediate also called hemifusion stalk, and progress then via a fusion pore (Chernomordik and Kozlov, 2008; Lang et al., 2008; Martens and McMahon, 2008). There is also compelling evidence that lipids directly participate in the different steps of the fusion process. For fusion to occur, membranes have to be into close proximity, and to be destabilized. It has been suggested that both events require the induction of membrane curvature at the fusion site (Martens and McMahon, 2008). Membrane curvature can be managed by specific proteins and peptidic sequences such as the C2 domains (Chernomordik and Kozlov, 2008; Hu et al., 2008; Martens and McMahon, 2008), but can also be highly provoked and regulated by lipids and their metabolism. The spontaneous curvature of lipids is related to their structure. PC and SM, which are cylindrical molecules, favor flat bilayer structures whereas lysophospholipids such as LPC and polyphosphoinositides such as PIP2 (inverted cone-shaped lipids) induce membranes with a positive curvature, and PA, PE, PS, DAG, ceramides (cone-shaped lipids) and fatty acids and cholesterol induce membranes with a

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negative curvature (Janmey and Kinnunen, 2006; Chernomordik and Kozlov, 2008). The stalk hypothesis considers that the formation of the hemifusion stalk intermediate is favored by lipids which induce negative curvature (Chernomordik and Kozlov, 2008; Lang et al., 2008). As a consequence, lipids inducing positive curvature such as LPC are inhibiting the early stages of membrane fusion whereas lipids inducing negative curvature such as PE are stimulating these steps (Chernomordik and Kozlov, 2008; Lang et al., 2008). Then the reverse is observed for the formation of the fusion pore, LPC favors and PE inhibits when they are added to the distal leaflets of the fusing membranes, suggesting that a positive curvature of the inner leaflets is required for full fusion to be achieved (Chernomordik and Kozlov, 2008). It has been suggested for example that PA stimulates the formation of the hemifusion state and PIP2 is required to complete fusion in the case of SNARE-driven liposome fusion (Vicogne et al., 2006). Cholesterol may help to drive vesicle fusion by virtue of its intrinsic negative curvature (Churchward et al., 2005). The sterol structure can affect membrane curvature and prepare a membrane for budding or fusion events (Bacia et al., 2005). Therefore, lipid metabolizing enzymes such as phospholipases, SMase, acyltransferases, and the overall lipid metabolism strongly contribute to membrane fusion and its regulation by providing lipids with specific structures compatible with the different steps of the fusion process. More and more examples are illustrating the requirement of such specific lipids in various intracellular fusion events (Janmey and Kinnunen, 2006; Brown et al., 2008; Chernomordik and Kozlov, 2008; Trajkovic et al., 2008; Yang et al., 2008 and references therein). In these phenomena, we have also to consider the cooperation between proteins and lipids in driving variations in membrane curvature (Yang et al., 2008). In addition, it has also to be mentioned that lipid phase separation, which may be handled by specific proteins, can trigger membrane fission (Roux et al., 2005). Finally, we have to consider that lipid metabolism (through phospholipases, acyltransferases, kinases, phosphatases, and the various metabolic pathways), can rapidly (from seconds to minutes) modify the membrane lipid composition in small or wider membrane domains.

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Table 3 Lipids demonstrated or expected to be required for mitochondrial membrane morphodynamics. Lipids

Physicochemical properties

Membrane effects and metabolism

Cer

Negative membrane curvature

OM/IM fusion/fission, microdomain formation Interactions with specific proteins?

CL/MLCL,

Negative membrane curvature

Specific interactions with OM proteins:

PG

Non lamellar phase inducers

OM permeabilization/apoptosis, OM fusion/fission Specific interactions with IM proteins: IM respiratory chain, IM fusion/fission

DAG

Negative membrane curvature Membrane dehydration, polar head spreading

OM/IM fusion/fission Regulation of enzymes of lipid metabolism

LPA

Inverted cone-shaped lipid

LPC

Positive membrane curvature

Decreases energy for membrane fusion OM/IM fusion/fission

PA

Negative membrane curvature

OM/IM fusion/fission Interactions with specific proteins?

PE

Negative membrane curvature Non lamellar phase inducer

OM/IM fusion/fission Regulated by PSD?

PIP2

Positive membrane curvature

?

PS

Negative membrane curvature Non lamellar phase inducer

OM/IM fusion/fission Regulated by PSD?

Cholesterol

Negative membrane curvature

OM/IM fusion/fission Stabilization of curved membranes

Cer: ceramides; DAG: diacylglycerol; IM: inner membranes; LCB: long chain bases; LPA, lyso-phosphatidic acid; LPC, lyso-phosphatidylcholine; OM: outer membranes; PSD: PS decarboxylase.

3.4. Lipid-assisted mitochondrial membrane dynamics A first thing to be considered about lipids in mitochondrial dynamics is that the submitochondrial membrane compartments have different lipid compositions, and therefore different physicochemical properties (Ardail et al., 1990). Beside the role of specific lipids in the function of the respiratory chain or apoptosis, such differences are probably also involved in the variations of membrane structures in relation to mitochondrial fusion. Recent data potentially implicate several lipid families as important factors in mitochondrial dynamics (Table 3). 3.4.1. CL and the other phospholipids CL is formed by the CL synthase through the condensation of PG with CDP-DAG to produce the diphosphatidylglycerol molecule which is CL. Interestingly, after its biosynthesis, CL undergoes remodeling of its fatty acyl chains with specific C18 unsaturated fatty acids (Gonzalvez and Gottlieb, 2007; Schlame, 2008). This was first believed to need the activity of a phospholipase A2 which generates a monolyso-CL (MLCL) which is then reacylated by a MLCL acyltransferase (Gonzalvez and Gottlieb, 2007; Joshi et al., 2009). This remodeling has been then attributed in animal cells to transacylation reactions governed by the tafazzin with PC as the acyl donor (Schlame, 2008 and references therein). The homolog yeast tafazzin has also been shown to function as an acyl-CoA independent LPC acyltransferase (Testet et al., 2005). The fact that yeast and human tafazzin-deficient cells accumulate MLCL may favor the first hypothesis but it has recently been proposed that the two pathways may be coupled in a more complex and intri-

cated scheme (see Fig. 8 in Schlame, 2008). CL is synthesized in the inner leaflet of the inner membrane and tafazzin was found to be located in the outer leaflet of the inner membrane and the inner leaflet of the outer membrane (Schlame, 2008). In addition, it has recently been discovered that a specific phospholipase D of the cytosolic surface of the outer membrane (MitoPLD) is hydrolyzing CL to produce PA, a negative curvature forming lipid that may facilitate fusion in a mitofusin-dependent manner (Choi et al., 2006). Therefore, CL must be translocated between membrane leaflets to be a substrate of these enzymes. Scramblase-3, creatine kinase and nucleoside diphosphate kinase may participate to CL movement between intramitochondrial membranes (Epand et al., 2007a,b; Schlame, 2008), and contact sites between outer and inner membranes may be critical for these processes (Piccotti et al., 2002). How CL, its precursor PG and its metabolite MLCL may be actors of mitochondrial fusion? First CL/MLCL and PG may form nonlamellar phases when they lose their negative charge, for example in the presence of Ca2+ ions (Epand et al., 2007a; Domonkos et al., 2008). The translocation of PG from one leaflet to the other one in outer or inner membranes may also destabilize the membranes (Devaux, 2000), and contribute to prepare membranes to fuse. In addition, clustering of CL by specific proteins may not only concentrate CL in domains, but also could induce phase separation and concentration in other domains of other lipids such as PE which is also a nonlamellar inducing molecule (Epand et al., 2007b). In addition, CL and PE are the major phospholipids of the mitochondrial con-

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tact sites between outer and inner membranes (Ardail et al., 1990; Gonzalvez and Gottlieb, 2007), in agreement with an involvement of these contact sites in mitochondria fusion processes. Finally, CL being the substrate of the MitoPLD which produces another fusogenic phospholipid (PA), CL metabolism and translocation between mitochondrial membranes may regulate the delivery of fusogenic lipids to different fusion sites. Moreover, the formation of PA may also recruit specific proteins of the fusion machinery as in the case of SNARE-dependent membrane fusion (Choi et al., 2006 and references therein). Another critical function of PA may be considered through the formation of its mono-acylated derivative LPA. Divalent ions such as Ca2+ can concentrate PA in domains which are then the target of phospholipases A to produce LPA (Faraudo and Travesset, 2007). PA is a cone-shaped lipid favoring negative membrane curvature, and LPA an inverted cone-shaped lipid which could decrease the energy needed for membrane fusion (Haucke and Di Paolo, 2007). Although the size of the polar head of LPA is smaller than that of LPC, we may consider that PA and LPA could play similar roles in the regulation of mitochondrial fusion as those demonstrated for PE and LPC in intracellular membrane fusion (see chapter III.3, Chernomordik and Kozlov, 2008). Of course PE and LPC may have also such actions in mitochondrial fusion. Finally, via an activation of PIP kinases, PA could also stimulate the biosynthesis of PIP2 species which have been shown to be critical in the function of the secretory pathway (Haucke and Di Paolo, 2007). However, it is not known whether phosphoinositides have a role in mitochondrial membrane dynamics. They may behave similarly to PA by forming PIP2 domains (Faraudo and Travesset, 2007), by destabilizing membrane structure (but in this case by inducing positive membrane curvature) and/or recruiting proteins of the fusion machinery and phospholipases such as phospholipase C which produces DAG (see below). Two other interesting phospholipids to be considered together are PE and PS. PS is synthesized by the PS synthase in mitochondrial-associated ER membranes and then transferred to the phosphatidylserine decarboxylase (PSD), which resides in the outer leaflet of the inner mitochondrial membrane and produces PE from PS. Beside the well-known role of PS at the cell-surface in apoptosis and in intracellular membrane trafficking (Haucke and Di Paolo, 2007), PS may also be another actor regulating fusion events in mitochondria. It is interesting to note that PS in the presence of Ca2+ is able to form nonlamellar phases as other phospholipids (CL, PA, PG) and PE which does not require Ca2+ . It has recently been observed that PSD could tightly control the ratios of PS/PE in mitochondrial membranes (Bellance, Furt, Molter, Melser, Letellier, Moreau, Rossignol; submitted), and therefore may adjust the concentrations of PS and PE to different tasks and submitochondrial compartments requiring nonlamellar-inducing lipids in the presence or not of Ca2+ . In this context, aminophospholipid translocases may also have crucial roles in participating to the delivery of these phospholipids to specific sites (Haucke and Di Paolo, 2007 and references therein). Finally, a balance between phospholipases and acyltransferases activities may participate in the regulation of the concentrations of specific lipids with different physicochemical properties in the zones of membrane fusion/fission. In this context it has to be noticed that endophilin B1 (a putative acyltransferase) is involved in mitochondrial morphology (Karbowski et al., 2004), and that LPC has a strong tendency to vesiculate membranes (Devaux, 2000). 3.4.2. Neutral lipids and sphingolipids Two neutral lipids have to be considered to be potential actors in mitochondrial dynamics, DAG and sterols. DAG has been clearly implicated in the regulation of intracellular membrane fusion events (see chapter III.2). DAG is an important metabolite for

phospholipid polymorphism, lipid signalling in microdomains via activation of protein kinase C for example, and can regulate numerous enzymes of phospholipid metabolism such as DAG kinase, phospholipases and CTP-phosphocholine cytidyltransferase (a key enzyme of PC biosynthesis) (Gomez-fernandez and CorbalanGarcia, 2007). DAG, by its tendency to favor negative membrane curvature, membrane dehydration and polar head spreading, may facilitate mitochondrial membrane fusion. In that respect, it is interesting to note that DAG is present in membrane contact sites and particularly in the inner membrane contact sites (Ardail et al., 1990). A balance between DAG and PA via regulation of DAG kinase and PA phosphatase may also be considered as a critical factor for managing mitochondrial membrane fusion. Therefore, by its action on several enzymes of phospholipid metabolism and its physisochemical properties, DAG might be another key actor of mitochondrial membrane dynamics. Ergosterol has been shown to be required in the maintenance of mitochondrial morphology in yeast (Altmann and Westermann, 2005). Behind the fact that sterols and therefore cholesterol are additional lipids which can induce negative membrane curvature and can shape membranes (Nomura et al., 2005), it has also to be considered that cholesterol may simply help to stabilize curved membrane microdomains (Haucke and Di Paolo, 2007 and references therein). Cholesterol has been found enriched in outer membrane contact sites (Ardail et al., 1990) which could also be related to a function in mitochondrial fusion. At this level, it is interesting to note that DAG is more concentrated in inner membrane contact sites and cholesterol in outer membrane contact sites, whether this feature is critical to mitochondrial dynamics will have to be determined. Sphingolipids are the last lipid family to be considered. PalmitoylCoA and serine associate to form long chain bases which are then matured in animal cells by addition of a phosphocholine polar head to synthesize sphingomyelin, or by addition of various sugars to make the different glycolipids up to the gangliosides. Interestingly, the biosynthesis of SM from ceramides (Cer) is using PC as the phosphocholine donor, and therefore liberates DAG. In addition, several SMase can generate Cer from SM, and LCB and Cer can be phosphorylated, and are involved together with Cer in lipid signalling (van Blitterswijk et al., 2003). As a consequence, sphingolipid metabolism will not only regulate Cer concentrations but also contribute to the regulation of DAG concentrations. DAG and Cer have similar structures and, as DAG, Cer favors negative membrane curvature. In addition, Cer has a packing effect on lipid bilayers through intermolecular hydrogen bonding and its more saturated fatty acyl chains. These properties lead Cer to segregate into microdomains. Cer has been implicated in the function of the secretory pathway (see chapter III.2) and in membrane permeability and channel formation (van Blitterswijk et al., 2003). What about Cer and mitochondria? It has been determined that Cer can be synthesized both in outer and inner mitochondrial membranes (Bionda et al., 2004). In addition, it has also been shown that Cer formed in the ER/mitochondria-associated ER membranes can be delivered to mitochondria (Stiban et al., 2008). A specific CERT-like protein (demonstrated to transport Cer in the ER-Golgi pathway), could deliver Cer to the mitochondria (Hanada, 2006). It can be suggested that both sources of Cer in outer membranes may contribute to regulate their fusion potential and permeabilization during apoptosis (van Blitterswijk et al., 2003; Stiban et al., 2008). Formation of Cer in the inner membranes may also be related to fusion/fission processes and to the function of the respiratory chain (Bionda et al., 2004 and references therein). Interestingly, Cer has been found to be even more concentrated in mitochondrial membranes than in endomembranes of the secretory pathway of basal epidermal cells, representing 48% of the total cell content

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of Cer (Vielhaber et al., 2001). These data strongly support the involvement of Cer in several critical functions of mitochondrial membranes as cited above. Cer can be hydrolyzed to LCB by the activity of ceramidases, which may be key partners of the regulation of Cer concentrations in several physiological functions (van Blitterswijk et al., 2003; Aerts et al., 2008). Raft-like microdomain formation with Cer and other sphingolipids such as the ganglioside GD3 may also contribute to the dynamics of mitochondria and their physiological functions (Malorni et al., 2007). Finally, it must be noticed that Cer, SM and cholesterol can have indirect regulatory roles on phospholipases A2 and D (Venable et al., 1996; Klapisz et al., 2000), which has also to be considered in the homeostasis of several lipidic metabolites involved in mitochondrial membrane dynamics. 4. Conclusions and future prospects It is more and more evidenced that through the outlines of lipid metabolism, the physicochemical properties of lipids and the specificities of lipids/lipids and lipids/proteins interactions, lipid polymorphism is a fundamental brick of membrane organisation and dynamics. This review has rapidly summarized how it is true for mitochondrial membranes and has highlighted how lipids and lipid-based machineries are or may be critical for the overall dynamics of mitochondria. Because mitochondrial membrane fusion/fission processes are clearly involved in mitochondria dynamics and that these events are undoubtedly critical for several cell functions and the balance cell life/cell death (Meeusen and Nunnari, 2005; Jensen and Sesaki, 2006; Detmer and Chan, 2007; Parone et al., 2008 and references therein), the challenge in the future will be to unravel the detailed mechanisms and to identify all the required partners. For this, the establishment of specific in vitro reconstitution systems and appropriate biochemical, genetic and imaging tools will be necessary. In addition, a better understanding of these processes in the context of human disorders (Detmer and Chan, 2007) may offer the possibility of new targets for pharmacological approaches. References Abousalham A, Hobman TC, Dewald J, Garbutt M, Brindley DN. Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor. Biochem J 2002;361:653–61. Aerts AM, Zabrocki P, Franc¸ois IE, Carmona-Gutierrez D, Govaert G, Mao C, et al. Ydc1p ceramidase triggers organelle fragmentation, apoptosis and accelerated ageing in yeast. Cell Mol Life Sci 2008;65:1933–42. Altmann K, Westermann B. Role of essential genes in mitochondrial morphogenesis in Saccharomyces cerevisiae. Mol Biol Cell 2005;16:5410–7. Ardail D, privat JP, Egret-Charlier M, Levrat C, lerme F, Louisot P. Mitochondrial contact sites: lipid composition and dynamics. J Biol Chem 1990;265:18797– 802. Bacia K, Schwille P, Kurzchalia T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc Natl Acad Sci 2005;102:3272–7. Bagnat M, Simons K. Cell surface polarization during yeast mating. J Biol Chem 2002;383:1475–80. Benard G, Bellance N, James D, Parrone P, Fernandez H, Letellier T, et al. Mitochondrial bioenergetics and structural network organization. J Cell Sci 2007;120:838–48. Benard G, Faustin B, Galinier A, Rocher C, Bellance N, Smolkova K, et al. Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases. Int J Biochem Cell Biol 2008;40:1543–54. Benard G, Rossignol R. Ultrastructure of the mitochondrion and its bearing on function and bioenergetics. Antioxid Redox Signal 2008;10:1313–42. Bigay J, Casella JF, Drin G, Mesmin B, Antonny B. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J 2005;24:2244–53. Bionda C, Portoukalian J, Schmitt D, Rodriguez-Lafrasse C, Ardail D. Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria? Biochem J 2004;382:527–33. Boldogh IR, Pon LA. Mitochondria on the move. Trends Cell Biol 2007;17:502–10.

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