RESUMO
Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.
Assuntos
Fusão de Membrana , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Vacúolos/metabolismoRESUMO
Yeast vacuolar HOPS tethers membranes, catalyzes trans-SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted-vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage. The MED fusion blockade is overcome by either salt extraction, inactivation with the MED ligand calmodulin, or addition of Sec17/Sec18. During incubation with MED, SNAREs assemble stable complexes in trans and fusion becomes resistant to antibody to the Qa SNARE. When Q-SNAREs are preassembled, a synthetic tether can replace HOPS for fusion. With a synthetic tether, fusion needs both complete SNARE zippering and Sec17/Sec18 to overcome a MED block. In contrast, when SNARE domains are only two-third zippered, only HOPS will support Sec17/Sec18 driven fusion without needing complete zippering. HOPS thus remains engaged with SNAREs during zippering. MED facilitates the study of distinct fusion stages: tethering, initial trans-SNARE assembly and its sensitivity to Sec17, SNARE zippering, Sec17/Sec18 engagement, and lipid and lumenal mixing.
Assuntos
Fusão de Membrana , Proteínas de Saccharomyces cerevisiae , Fusão de Membrana/fisiologia , Proteínas de Transporte Vesicular , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Ligantes , Proteínas SNARE , Saccharomyces cerevisiae/fisiologia , Vacúolos , Lipídeos , Proteínas Q-SNARERESUMO
As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the two membranes together. Because Qa- and Qb-SNAREs are anchored to the same membrane and are adjacent in the 4-SNARE bundle, their two anchors might be redundant. Using the recombinant pure protein catalysts of yeast vacuole fusion, we now report that the specific distribution of transmembrane (TM) anchors on the Q-SNAREs is critical for efficient fusion. A TM anchor on the Qa-SNARE supports rapid fusion even when the other two Q-SNAREs are unanchored, while a TM anchor on the Qb-SNARE is dispensable and is insufficient for rapid fusion as the sole Q-SNARE anchor. This does not depend on which specific TM domain is attached to the Qa-SNARE but rather is due to the Qa-SNARE being anchored per se. The need for Qa-SNARE anchoring is even seen when the homotypic fusion and vacuole protein sorting protein (HOPS), the physiological catalyst of tethering and SNARE assembly, is replaced by an artificial tether. The need for a Qa TM anchor is thus a fundamental property of vacuolar SNARE zippering-induced fusion and may reflect the need for the Qa juxtamembrane (JxQa) region to be anchored between its SNARE and TM domains. This requirement for Qa-SNARE anchoring and correct JxQa position is bypassed by Sec17/Sec18, exploiting a platform of partially zippered SNAREs. Because Qa is the only synaptic Q-SNARE with a TM anchor, the need for Qa-specific anchoring may reflect a general requirement for SNARE-mediated fusion.
Assuntos
Proteínas de Saccharomyces cerevisiae , Vacúolos , Vacúolos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Q-SNARE/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa, Qb, and Qc families, and SNARE chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. Yeast vacuole fusion is regulated by phosphatidylinositol-3-phosphate (PI3P). PI3P binds directly to the vacuolar Qc-SNARE and to HOPS, the vacuolar tethering/SM complex. We now report several distinct functions of PI3P in fusion. PI3P binds the N-terminal PX domain of the Qc-SNARE to enhance its engagement for fusion. Even when Qc has been preassembled with the Qa- and Qb-SNAREs, PI3P still promotes trans-SNARE assembly and fusion between these 3Q proteoliposomes and those with R-SNARE, whether with the natural HOPS tether or with a synthetic tether. With HOPS, efficient trans-SNARE complex formation needs PI3P on the 3Q-SNARE proteoliposomes, in cis to the Qc. PI3P is also needed for HOPS to confer resistance to Sec17/Sec18. With a synthetic tether, fusion is supported by PI3P on either fusion partner membrane, but this fusion is blocked by Sec17/Sec18. PI3P thus supports multiple stages of fusion: the engagement of the Qc-SNARE, trans-SNARE complex formation with preassembled Q-SNAREs, HOPS protection of SNARE complexes from Sec17/Sec18, and fusion per se after tethering and Q-SNARE assembly.
Assuntos
Fusão de Membrana , Proteínas de Saccharomyces cerevisiae , Humanos , Adenosina Trifosfatases/metabolismo , Fusão de Membrana/fisiologia , Proteínas Qc-SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Membrane fusion is driven by Sec17, Sec18, and SNARE zippering. Sec17 bound to SNAREs promotes fusion through its membrane-proximal N-terminal apolar loop domain. At its membrane-distal end, Sec17 serves as a high-affinity receptor for Sec18. At that distance from the fusion site, it has been unclear how Sec18 can aid Sec17 to promote fusion. We now report that Sec18, with ATPγS, lowers the Km of Sec17 for fusion. A C-terminal and membrane-distal Sec17 mutation, L291A,L292A, diminishes Sec17 affinity for Sec18. High levels of wild-type Sec17 or Sec17-L291AL292A show equivalent fusion without Sec18, but Sec18 causes far less fusion enhancement with low levels of Sec17-L291AL292A than with wild-type Sec17. Another mutant, Sec17-F21SM22S, has reduced N-loop apolarity. Only very high levels of this mutant protein support fusion, but Sec18 still lowers the apparent fusion Km for Sec17-F21SM22S. Thus Sec18 stimulates fusion through Sec17 and acts at the well-described interface between Sec18 and Sec17. ATP acts as a ligand to activate Sec18 for Sec17-dependent fusion, but ATP hydrolysis is not required. Even without SNAREs, Sec18 and Sec17 exhibit interdependent stable association with lipids, with several Sec17 bound for each Sec18 hexamer, explaining how Sec18 stabilization of surface-concentrated clusters of Sec17 lowers the Sec17 Km for assembly with SNAREs. Each of the associations, between SNARE complex, Sec18, Sec17, and lipid, helps assemble the fusion machinery.
Assuntos
Fusão de Membrana , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Ligantes , Lipídeos , Fusão de Membrana/fisiologia , Proteínas Mutantes/metabolismo , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Membrane fusion requires tethers, SNAREs of R, Qa, Qb, and Qc families, and chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. SNAREs have N-domains, SNARE domains that zipper into 4-helical RQaQbQc coiled coils, a short juxtamembrane (Jx) domain, and (often) a C-terminal transmembrane anchor. We reconstitute fusion with purified components from yeast vacuoles, where the HOPS protein combines tethering and SM functions. The vacuolar Rab, lipids, and R-SNARE activate HOPS to bind Q-SNAREs and catalyze trans-SNARE associations. With SNAREs initially disassembled, as they are on the organelle, we now report that R- and Qa-SNAREs require their physiological juxtamembrane (Jx) regions for fusion. Swap of the Jx domain between the R- and Qa-SNAREs blocks fusion after SNARE association in trans. This block is bypassed by either Sec17, which drives fusion without requiring complete SNARE zippering, or transmembrane-anchored Qb-SNARE in complex with Qa. The abundance of the trans-SNARE complex is not the sole fusion determinant, as it is unaltered by Sec17, Jx swap, or the Qb-transmembrane anchor. The sensitivity of fusion to Jx swap in the absence of a Qb transmembrane anchor is inherent to the SNAREs, because it remains when a synthetic tether replaces HOPS.
Assuntos
Proteínas SNARE , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/metabolismo , Fusão de Membrana/fisiologia , Proteolipídeos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.
Assuntos
Adenosina Trifosfatases/metabolismo , Fusão de Membrana/genética , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/genética , Comunicação Celular , Fusão de Membrana/fisiologia , Domínios Proteicos , Proteínas SNARE/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Yeast vacuole fusion requires R-SNARE, Q-SNAREs, and HOPS. A HOPS SM-family subunit binds the R- and Qa-SNAREs. We now report that HOPS binds each of the four SNAREs. HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled, ushering them into a functional complex. Co-incubation of HOPS, proteoliposomes bearing R-SNARE, and proteoliposomes with any two Q-SNAREs yields a rapid-fusion complex with 3 SNAREs in a trans-assembly. The missing Q-SNARE then induces sudden fusion. HOPS can 'template' SNARE complex assembly through SM recognition of R- and Qa-SNAREs. Though the Qa-SNARE is essential for spontaneous SNARE assembly, HOPS also assembles a rapid-fusion complex between R- and QbQc-SNARE proteoliposomes in the absence of Qa-SNARE, awaiting Qa for fusion. HOPS-dependent fusion is saturable at low concentrations of each Q-SNARE, showing binding site functionality. HOPS thus tethers membranes and recognizes each SNARE, assembling R+Qa or R+QbQc rapid fusion intermediates.
Assuntos
Fusão de Membrana , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Membranas Intracelulares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.
Assuntos
Adenosina Trifosfatases/metabolismo , Fusão de Membrana , Proteolipídeos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/química , Transporte Biológico , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Proteolipídeos/química , Proteínas SNARE/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/químicaRESUMO
Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.
Assuntos
Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Citoplasma/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana , Lipídeos de Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas SNARE/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/química , Bicamadas Lipídicas/química , Lipídeos/química , Lipossomos/química , Fusão de Membrana , Mutação , Ligação Proteica , Proteolipídeos/química , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Lipídeos de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Caseína Quinase I/metabolismo , Guanosina Trifosfato/metabolismo , Immunoblotting , Lipossomos/química , Lipossomos/metabolismo , Fusão de Membrana , Lipídeos de Membrana/química , Fosforilação , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
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.