[44] Methods for studying the yeast vacuole

Roberts, Christopher J.; Raymond, Christopher K.; Yamashiro, Carl T.; Stevens, Tom H.

doi:10.1016/0076-6879(91)94047-g

Cited by 331 publications

(309 citation statements)

References 45 publications

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“…An additional test was performed to specifically identify disruptants impaired in vacuolar targeting via the CPY pathway. This test was based on colony immunoblotting to detect the secretion of CPY, a sorting defect exhibited by a subset of endocytic (end) and vacuolar protein sorting (vps) mutants (Munn and Riezman, 1994;Roberts et al, 1991). We identified 25 mutants among the 631 viable deletion strains included in our screens, that correspond to unknown genes or genes recently identified as being involved in trafficking or glycosylation.…”

Section: Eurofan Deletants Impaired In Secretion and Vacuolar Trafficmentioning

confidence: 99%

“…The filter was then removed from the plate, the cells washed off with water and we tested for immunodetectable CPY, as described above (Roberts et al, 1991). All the tests included the following controls: both wild-type strains, and two mutant strains, vps1 (Rothman et al, 1990), defective in a dynamin-like GTPase and vps4/end13 (Babst et al, 1998;Munn and Riezman, 1994;Zahn et al, 2001), defective in an AAA-ATPase, both proteins required for vacuolar protein sorting and both mutants displaying CPY secretion (Munn and Riezman, 1994).…”

Section: Eurofan Deletants Impaired In Secretion and Vacuolar Trafficmentioning

confidence: 99%

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Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

Avaro

Belgareh‐Touzé

Sibella-Argüelles

et al. 2002

Yeast

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We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble a-factor, the periplasmic glycoprotein invertase, the plasma membrane GPIanchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.

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Section: Eurofan Deletants Impaired In Secretion and Vacuolar Trafficmentioning

confidence: 99%

Section: Eurofan Deletants Impaired In Secretion and Vacuolar Trafficmentioning

confidence: 99%

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

Avaro

Belgareh‐Touzé

Sibella-Argüelles

et al. 2002

Yeast

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“…Additional mutants were obtained by transforming the same strains with a NotI-digested yeast genomic library containing random insertions of a Tn3-LacZ transposon cassette (Burns et al, 1994). CPY-secreting colonies were identified by an overlay assay (Roberts et al, 1991), and those that exhibited synthetic lethality with end4⌬ (as determined by the inability to grow in the presence of 5-FOA) were chosen for further study. Mutants were tested for complementation with each other and with the existing vps mutant collection.…”

Section: Yeast Genetic Screenmentioning

confidence: 99%

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

Conibear

Stevens

2000

MBoC

Self Cite

266

336

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The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/ prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.

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“…To characterize whether VRP1 deletion and upregulation of ERG6 affects vacuolar protein sorting, we examined if CpY, a vacuolar luminal protein, was missorted to the plasma membrane by filter binding assays (Roberts et al, 1991). Starting with cultures at 1 OD 600 , a 10-fold dilution series was plated onto YP-agar with either 2% dextrose or 1% galactose/0.2% dextrose to induce ERG6 expression.…”

Section: Vol 15 October 2004mentioning

confidence: 99%

“…The amount of precipitated protein was determined by Bradford assay and expressed as a percentage of total in Table 3. Extracellular CpY was analyzed by secretion filter binding assay as described (Roberts et al, 1991). Briefly, 3 l of a 10-fold dilution series was plated onto YPD or 1% galactose/0.2% dextrose (YPG), overlaid with nitrocellulose filters, incubated for 48 h at 28°C, and then immunoblotted for CpY.…”

Section: Protein Processing and Analysismentioning

confidence: 99%

Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

Tedrick

Trischuk

Lehner

et al. 2004

MBoC

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Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1⌬ growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1⌬ mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1⌬ vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1⌬ strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1⌬-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1⌬ strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.

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[44] Methods for studying the yeast vacuole

Cited by 331 publications

References 45 publications

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

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