G-protein ligands inhibit in vitro reactions of vacuole inheritance.

Haas, Albert; Conradt, Barbara; Wickner, William

doi:10.1083/jcb.126.1.87

Cited by 168 publications

(212 citation statements)

References 86 publications

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“…Thus the ␤-lactamase assay measures authentic vacuole fusion. However, microcystin-LR (MCLR) and the nonhydrolyzable GTP analogue GTP␥S, previously reported as fusion reaction inhibitors (12,13), showed different inhibitory effects ( Fig. 1 D and E).…”

Section: Resultsmentioning

confidence: 99%

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Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Jun

Wickner

2007

Proc. Natl. Acad. Sci. U.S.A.

111

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Membrane fusion entails organelle docking and subsequent mixing of membrane bilayers and luminal compartments. We now present an in vitro assay of fusion, using yeast vacuoles bearing domains of either Fos or Jun fused to complementary halves of ␤-lactamase. Upon fusion, these proteins associate to yield ␤-lactamase activity. This assay complements the standard fusion assay (activation of pro-Pho8p in protease-deficient vacuoles by proteases from pho8⌬ vacuoles). Both the ␤-lactamase and proPho8p activation assays of fusion show the same long kinetic delay between SNARE pairing and luminal compartment mixing. Lipidmixing occurs rapidly after SNARE pairing but well before aqueous compartment mixing. These results support a model in which SNARE pairing leads to rapid hemifusion, followed by slow further lipid rearrangement and aqueous compartment mixing. (diacylglycerol, phosphoinositides, and ergosterol) are concentrated at a ring-shaped microdomain, the ''vertex ring,'' surrounding the apposed membranes of docked vacuoles. During docking, the released Vam7p rebinds to the vacuole through its affinities for phosphatidylinositol 3-phosphate (1) and HOPS (2) and participates in the formation of trans-SNARE complexes between apposed vacuolar membranes. Bilayer fusion around the vertex ring permits lipid and aqueous compartment mixing. It has been proposed that membrane fusion may require a lipidic ''hemifusion'' intermediate, where the closely apposed membrane leaflets are fused and mix, whereas the distal leaflets and aqueous compartments remain distinct. Hemifusion has been directly observed with influenza HA (3-5), either wild-type or with a GPI-anchor in place of its transmembrane domain (TMD), in a minimal SNARE-driven liposome fusion system (6, 7) with low levels of wild-type SNARE proteins or with mutant SNARE proteins with partial TMDs that span only one bilayer leaflet (6). Hemifusion was also observed in cell-cell fusion mediated by GPI-anchored SNARE proteins expressed on the outer surface of the plasma membrane (8), suggesting that all SNARE-dependent fusion might proceed by means of hemifusion. A hemifusion intermediate was proposed for yeast vacuole fusion, based on the finding that GTP␥S allows lipid mixing while blocking the fusion-dependent activation of pro-Pho8p (9). The standard in vitro assay of vacuole fusion measures the proteolytic activation of the inactive proPho8p (alkaline phosphatase or ALP) by vacuolar proteases upon content mixing between two populations of vacuoles. One limitation of this assay is its sensitivity to reagents that inhibit the activation of ALP. We have therefore developed an in vitro assay of yeast vacuole fusion in which content mixing between two vacuole populations is measured by the reconstitution of active Escherichia coli TEM-1 ␤-lactamase from two complementary fragments of the enzyme, each contributed from distinct vacuole populations. This fusion assay, coupled with a lipid mixing assay, has revealed that docking and lipid mixing are completed long before l...

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Section: Resultsmentioning

confidence: 99%

“…Fos-or BJ3505-␣-Jun (13). Standard ␤-lactamase fusion reactions (60 l) contained 6 g of vacuoles containing the fusion protein Fos-, 6 g of vacuoles with ␣-Jun, the same reaction buffer, ATP, creatine phosphate, creatine kinase, and CoA as for the ALP fusion assay, and 11 M recombinant GST-Fos protein (to reduce any background signal due to lysis).…”

mentioning

confidence: 99%

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Jun

Wickner

2007

Proc. Natl. Acad. Sci. U.S.A.

111

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“…Strains-The strains used for fusion assays were BJ3505 (MAT␣ pep4::HIS3 prb1-⌬1.6R his3-200 lys2-801 trp1⌬101 (gal3) ura3-52 gal2 can1) (38) and DKY6281 (MAT␣ leu2-3 leu 2-112 ura3-52 his3-⌬200 trp1-⌬901 lys2-801) (16). BJ3505 nyv1⌬ and DKY6281 nyv1⌬ were described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…Vacuoles fission and fuse during cell division and organelle inheritance (14) and in response to growth medium osmolarity (15). Purified vacuoles undergo a multistep fusion pathway, which can be monitored by content-mixing assays (16). In the initial "priming" step, Sec18p/Sec17p disassemble cis-SNARE complexes (17).…”

mentioning

confidence: 99%

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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SNARE proteins form bundles of four ␣-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q⅐1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7 Q283R , yet Vam7 Q283R is preferred by vacuoles with Nyv1 R191Q . Rotation of the position of the arginine in the 0-layer increases the K m for Vam7p but does not affect the maximal rate of fusion. Vam7 Q283R forms stable 2Q⅐2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion. SNARE 4 proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/␣-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/␣-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impress...

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“…We therefore analyzed the role of Pfa3 in in vitro vacuole fusion. We deleted PFA3 from both tester strains and used them in the fusion reaction (30). Vacuoles lacking Pfa3 showed a 50% reduction in fusion (Fig.…”

Section: Pfa3 Affects Vacuole Fusion and Vac8mentioning

confidence: 99%

The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8

Hou

Subramanian

LaGrassa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3͞Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuoleassociated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate͞palmitate (as in Vac8) but also one that is targeted by a myristate͞basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.Yck3 ͉ SH4 domain ͉ acylation ͉ membrane targeting P rotein and lipid trafficking along the endomembrane system occurs by vesicular transport (1). Of all proteins implicated in vesicle fusion and fission, only a subset is permanently associated with membranes via transmembrane segments, whereas most are recruited to the membrane from the cytoplasm. The latter proteins depend on membrane receptors, lipids, or lipid anchors for binding to their appropriate target membrane (2). This poses the question of how the recruitment of these proteins is coordinated with their function.Palmitoylation has been discussed as a special lipid modification. It may direct proteins to specific membrane domains (3-5), and it is the only common lipid modification that is reversible, permitting cycling of a protein between membranes and cytosol. The identification and characterization of the underlying acylation͞deacylation machinery is therefore critical for understanding the function of palmitoylation. Recently, biochemical and genetic analyses have identified several proteins that are required for palmitoylation, including those of the so-called DHHC-CRD family of polytopic membrane proteins (6). Yeast contains seven homologs (Erf2, Swf1, Yol003c, Akr1, Akr2, Ydr459c, and Ynl326c͞Pfa3), which seem to be distributed throughout the endomembrane system as suggested by the GFP database and recent studies. At the ER, Swf1 is required for palmitoylation of Tlg1 (7), and Erf2 promotes Ras2 palmitoylation (8, 9). The Golgi-localized Akr1 is responsible for palmitoylation of the casein kinase I (CKI) isoform Yck2 (10). Similarly, several of the 20 or so mammalian DHHC proteins localize to distinct organelles, with critical roles for the palmitoylation of PSD-95, Ras, and SNAP-25 (11-13). This wide distribution of proteins involved in palmitoylation suggests that palmitoylati...

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G-protein ligands inhibit in vitro reactions of vacuole inheritance.

Cited by 168 publications

References 86 publications

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8

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