Yeast homotypic vacuole fusion requires the Ccz1–Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.

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The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting–Class C Vps complex

Biochemical Journal ◽

10.1042/bj20110687 ◽

2012 ◽

Vol 443 (1) ◽

pp. 205-211 ◽

Cited By ~ 5

Author(s):

Christopher Stroupe

Keyword(s):

Protein Sorting ◽

Rab Gtpase ◽

Main Function ◽

Rab Gtpases ◽

Membrane Recruitment ◽

Vacuole Fusion ◽

Protein Receptor ◽

Head Groups ◽

Class C ◽

Hops Complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)–Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3′-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity–that is, beyond bulk effector recruitment–for a Rab GTPase.

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Phosphatidylinositol 3,5-bisphosphate regulates the transition between trans-SNARE complex formation and vacuole membrane fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e18-08-0505 ◽

2019 ◽

Vol 30 (2) ◽

pp. 201-208 ◽

Cited By ~ 8

Author(s):

Gregory E. Miner ◽

Katherine D. Sullivan ◽

Annie Guo ◽

Brandon C. Jones ◽

Logan R. Hurst ◽

...

Keyword(s):

Complex Formation ◽

Membrane Fusion ◽

Molecular Switch ◽

Snare Complex ◽

Cellular Functions ◽

Vacuole Membrane ◽

Hyperosmotic Shock ◽

Outer Leaflet ◽

Vacuole Fusion ◽

Snare Complex Formation

Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion and fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after trans-SNARE pairing. Although SNARE complex formation was unaffected, we found that C8-PI(3,5)P2 blocked outer leaflet lipid mixing. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited the lipid mixing stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present at elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

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The N-terminal Domain of the t-SNARE Vam3p Coordinates Priming and Docking in Yeast Vacuole Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3375 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3375-3385 ◽

Cited By ~ 42

Author(s):

Rico Laage ◽

Christian Ungermann

Keyword(s):

Membrane Fusion ◽

Terminal Region ◽

Snare Complex ◽

Truncated Protein ◽

Sequence Of Events ◽

Vacuole Fusion ◽

N Terminus ◽

Hops Complex

Homotypic fusion of yeast vacuoles requires a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with thecis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3ΔN) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of thecis-SNARE complex is more efficient on Vam3ΔN vacuoles; however, the HOPS complex is not associated well with the Vam3ΔN cis-complex. Thus, primed SNAREs from Vam3ΔN vacuoles cannot participate efficiently in the reaction becausetrans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.

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HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0077 ◽

2008 ◽

Vol 19 (6) ◽

pp. 2500-2508 ◽

Cited By ~ 92

Author(s):

Vincent J. Starai ◽

Christopher M. Hickey ◽

William Wickner

Keyword(s):

Snare Complex ◽

Wild Type ◽

Fusion Event ◽

Sm Proteins ◽

Terminal Domain ◽

Domain Structures ◽

Vacuole Fusion ◽

Protein Receptors ◽

Optimal Fusion ◽

Guanosine Triphosphatase

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Km value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.

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A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

The Journal of Cell Biology ◽

10.1083/jcb.140.1.61 ◽

1998 ◽

Vol 140 (1) ◽

pp. 61-69 ◽

Cited By ~ 179

Author(s):

Christian Ungermann ◽

Benjamin J. Nichols ◽

Hugh R.B. Pelham ◽

William Wickner

Keyword(s):

Atp Hydrolysis ◽

Complex Structure ◽

Snare Complex ◽

Functional Aspect ◽

Independent Function ◽

Attachment Protein ◽

Vacuole Fusion ◽

Predominant Form ◽

Target Membrane

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

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