ABSTRACT
Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here, we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 (NSF) catalysis, Sec17 (α-SNAP) either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, Vps33/HOPS promotes productive SNARE assembly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially zipped, Sec17 promotes fusion in either the presence or absence of HOPS, but with faster kinetics when HOPS is absent, suggesting that ejection of the SM is a rate-limiting step.
Subject(s)
Intracellular Membranes/physiology , Membrane Fusion , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.
Subject(s)
SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , rab GTP-Binding Proteins/metabolism , Membrane Fusion/physiology , Membrane Proteins/metabolism , Protein Binding , Proteolipids/genetics , Proteolipids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
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 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.
Subject(s)
Munc18 Proteins/metabolism , Qa-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism , Crystallography, X-Ray , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/ultrastructure , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/ultrastructureABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Membrane/metabolism , Gene Expression Regulation, Fungal , SNARE Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolism , Adenosine Triphosphatases/chemistry , Lipid Bilayers/chemistry , Lipids/chemistry , Liposomes/chemistry , Membrane Fusion , Mutation , Protein Binding , Proteolipids/chemistry , Saccharomyces cerevisiae/metabolismABSTRACT
Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.
Subject(s)
Membrane Lipids/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Casein Kinase I/metabolism , Guanosine Triphosphate/metabolism , Immunoblotting , Liposomes/chemistry , Liposomes/metabolism , Membrane Fusion , Membrane Lipids/chemistry , Phosphorylation , Protein Binding , Protein Subunits/metabolism , Qa-SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.
Subject(s)
Membrane Fusion , Vacuoles/metabolism , Biomarkers/metabolism , Fluorescent Dyes/metabolism , Inositol Phosphates/metabolism , Lipids/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolismABSTRACT
Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.
Subject(s)
Membrane Fusion , Membrane Lipids/metabolism , Membranes, Artificial , Q-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Vacuoles/metabolism , Adenosine Triphosphatases/metabolism , Binding Sites , Membrane Lipids/chemistry , Molecular Structure , Protein Binding , Protein Conformation , Proteolipids/metabolism , Q-SNARE Proteins/chemistry , R-SNARE Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Time Factors , Vacuoles/chemistry , Vesicular Transport Proteins/metabolismABSTRACT
The fusion of yeast vacuolar membranes depends on the disassembly of cis-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.
Subject(s)
Biocatalysis , Multiprotein Complexes/metabolism , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Biotinylation , Inositol Phosphates/metabolism , Solubility , Streptavidin/metabolismABSTRACT
The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle-Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)-this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.
Subject(s)
Casein Kinase I/metabolism , Cell Membrane/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolism , Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Phosphorylation , Protein Sorting Signals , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolism , ras GTPase-Activating Proteins/metabolismABSTRACT
The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-D-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.
Subject(s)
Membrane Fusion , Molecular Chaperones/metabolism , SNARE Proteins/metabolism , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolism , Adenosine Triphosphatases/metabolism , Biotin/pharmacokinetics , Cell Compartmentation , Dithionite/pharmacokinetics , Hymecromone/analogs & derivatives , Hymecromone/pharmacokinetics , Intracellular Membranes , Lipids/chemistry , Permeability , Protein Transport , Proteolipids/chemistry , Proteolipids/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.
Subject(s)
Membrane Fusion/physiology , SNARE Proteins/physiology , Lipid-Linked Proteins/chemistry , Lipid-Linked Proteins/physiology , Membrane Proteins/chemistry , Membrane Proteins/physiology , Protein Multimerization , Proteolipids/chemistry , Proteolipids/physiology , Q-SNARE Proteins/chemistry , Q-SNARE Proteins/physiology , R-SNARE Proteins/chemistry , R-SNARE Proteins/physiology , Recombinant Proteins/chemistry , SNARE Proteins/chemistry , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Vacuoles/chemistry , Vacuoles/physiologyABSTRACT
Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.
Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Serine Endopeptidases/metabolism , Serine Proteases/metabolism , Amino Acid Sequence , Binding Sites/genetics , Conserved Sequence , GTP-Binding Proteins/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Membrane Proteins/metabolism , Metalloproteases/metabolism , Mitochondrial Proteins/genetics , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Species Specificity , Substrate SpecificityABSTRACT
The mitochondrial dynamin-like GTPase Mgm1 exists as a long (l-Mgm1) and a short isoform (s-Mgm1). They both are essential for mitochondrial fusion. Here we show that the isoforms interact in a homotypic and heterotypic manner. Their submitochondrial distribution between inner boundary membrane and cristae was markedly different. Overexpression of l-Mgm1 exerts a dominant negative effect on mitochondrial fusion. A functional GTPase domain is required only in s-Mgm1 but not in l-Mgm1. We propose that l-Mgm1 acts primarily as an anchor in the inner membrane that in concert with the GTPase activity of s-Mgm1 mediates the fusion of inner membranes.
Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Membrane Fusion/physiology , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Dynamins/chemistry , Dynamins/metabolism , Microscopy, Immunoelectron , Mitochondrial Proteins/genetics , Protein Isoforms/metabolismABSTRACT
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.
Subject(s)
Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membranes/physiology , Apoptosis , Energy Metabolism , Humans , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Models, Biological , Parkinson Disease/metabolismABSTRACT
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.
Subject(s)
Mitochondria/enzymology , Mitochondrial Diseases/enzymology , Mitochondrial Proton-Translocating ATPases/genetics , Animals , DNA, Mitochondrial/metabolism , Humans , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/therapy , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolism , Models, Biological , Mutation , Protein Subunits/genetics , Protein Subunits/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription, GeneticABSTRACT
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.