Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast vacuolar homotypic fusion and vacuole protein sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.

Mitochondria are double-membrane enclosed eukaryotic organelles with a central role in numerous cellular functions. The ultrastructure of mitochondria varies considerably between tissues, organisms, and the physiological state of cells. Alterations and remodeling of inner membrane structures are evident in numerous human disorders and during apoptosis. The inner membrane is composed of two subcompartments, the cristae membrane and the inner boundary membrane. Recent advances in electron tomography led to the current view that these membrane domains are connected by rather small tubular structures, termed crista junctions. They have been proposed to regulate the dynamic distribution of proteins and lipids as well as of soluble metabolites between individual mitochondrial subcompartments. One example is the release of cytochrome c upon induction of apoptosis. However, only little is known on the molecular mechanisms mediating the formation and maintenance of cristae and crista junctions. Here we review the current knowledge of the factors that determine cristae morphology and how the latter is linked to mitochondrial function. Further, we formulate several theoretical models which could account for the de novo formation of cristae as well as their propagation from existing cristae.

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria. INTRODUCTIONMitochondria form large networks of interconnected tubules that are maintained by balanced fission and fusion events (Nunnari et al., 1997;Okamoto and Shaw, 2005). The morphology and ultrastructure of mitochondria depend on the tissue, on the physiological condition of the cell, and, in particular, on the functional status of mitochondria. Dynamic processes associated with mitochondria are apparently crucial for the cell, e.g., in apoptosis (Frank et al., 2001;Karbowski et al., 2002;Lee et al., 2004;Jagasia et al., 2005). Likewise, formation of dendritic spines and synapses (Li et al., 2004;Verstreken et al., 2005) and functional complementation of mitochondrial DNA (mtDNA) mutations by content mixing Ono et al., 2001) depend on dynamics of mitochondria. In contrast, vast morphological alterations of mitochondria have been reported to occur in human disorders. Impairment of mitochondrial fusion is causative of neurodegenerative diseases such as CharcotMarie-Tooth disease type 2A and 4A, and optic atrophy type 1 (Alexander et al., 2000;Delettre et al., 2000;Zuchner et al., 2004;Niemann et al., 2005).A key player in regulating mitochondrial fusion is the dynamin-like GTPase OPA1 (Olichon et al., 2003;Cipolat et al., 2004). Mutations in the OPA1 gene cause autosomal dominant optic atrophy type I, a prevalent hereditary neuropathy of the optic nerve (Alexander et al., 2000;Delettre et al., 2000). Down-regulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction, altered maintenance of mtDNA, altered mitochondrial inner membrane morphology, and increased propensity for apoptosis (Olichon et al., 2003;Griparic ...

Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP-or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17 L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17 F21S,M22S , with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17 F21S,M22S , interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.I ntracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and "effector" proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can "snare" (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved "SNARE domain" with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis-or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimidesensitive factor), an AAA family ATPase that drives SNARE c...

In mammals, the majority of cellular ATP is produced by the mitochondrial F1F(O)-ATP synthase through an elaborate catalytic mechanism. While most subunits of this enzymatic complex are encoded by the nuclear genome, a few essential components are encoded in the mitochondrial genome. The biogenesis of this multi-subunit enzyme is a sophisticated multi-step process that is regulated on levels of transcription, translation and assembly. Defects that result in diminished abundance or functional impairment of the F1F(O)-ATP synthase can cause a variety of severe neuromuscular disorders. Underlying mutations have been identified in both the nuclear and the mitochondrial DNA. The pathogenic mechanisms are only partially understood. Currently, the therapeutic options are extremely limited. Alternative methods of treatment have however been proposed, but still encounter several technical difficulties. The application of novel scientific approaches promises to deepen our understanding of the molecular mechanisms of the ATP synthase, unravel novel therapeutic pathways and improve the unfortunate situation of the patients suffering from such diseases.