Fusion of Docked Membranes Requires the Armadillo Repeat Protein Vac8p

Wang, Yong Xu; Kauffman, Emily J.; Duex, Jason E.; Weisman, Lois S.

doi:10.1074/jbc.m103937200

Cited by 83 publications

(91 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Mutation of all three cysteine residues to serine produces a Vac8 protein that is no longer palmitoylated in vivo (18) or in vitro by Pfa3 (26). The mutation also produces several phenotypes, including defective vacuolar inheritance (18), defective homotypic vacuole fusion in vitro, and fragmentation of the vacuole in vivo (24).…”

Section: Vac8 Is Palmitoylated By Pfa3 At All Three N-terminal Cysteimentioning

confidence: 99%

“…Vac8 is a scaffolding protein consisting primarily of 11 armadillo repeats involved in protein-protein interactions (19,20). Located at the vacuolar limiting membrane, Vac8 is involved in several membrane-mediated events, including vacuolar fusion, vacuolar inheritance, cytoplasm-tovacuole targeting, and nuclear autophagy (18,(21)(22)(23)(24). The N-terminal cysteines, and presumably the palmitoylation that occurs there, are required for the function of Vac8 in vacuolar fusion and vacuolar inheritance (18,24,25).…”

mentioning

confidence: 99%

“…Located at the vacuolar limiting membrane, Vac8 is involved in several membrane-mediated events, including vacuolar fusion, vacuolar inheritance, cytoplasm-tovacuole targeting, and nuclear autophagy (18,(21)(22)(23)(24). The N-terminal cysteines, and presumably the palmitoylation that occurs there, are required for the function of Vac8 in vacuolar fusion and vacuolar inheritance (18,24,25). We recently demonstrated both genetically and biochemically that the yeast DHHC protein Pfa3 is a Vac8 PAT (26).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Nadolski¹,

Linder

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Palmitoylation of the yeast vacuolar protein Vac8 is important for its role in membrane-mediated events such as vacuole fusion. It has been established both in vivo and in vitro that Vac8 is palmitoylated by the Asp-His-His-Cys (DHHC) protein Pfa3. However, the determinants of Vac8 critical for recognition by Pfa3 have yet to be elucidated. This is of particular importance because of the lack of a consensus sequence for palmitoylation. Here we show that Pfa3 was capable of palmitoylating each of the three N-terminal cysteines of Vac8 and that this reaction was most efficient when Vac8 is N-myristoylated. Additionally, when we analyzed the Src homology 4 (SH4) domain of Vac8 independent of the rest of the protein, palmitoylation by Pfa3 still occurred. However, the specificity of palmitoylation seen for the full-length protein was lost, and the SH4 domain was palmitoylated by all five of the yeast DHHC proteins tested. These data suggested that a region of the protein C-terminal to the SH4 domain was important for conferring specificity of palmitoylation. This was confirmed by use of a chimeric protein in which the SH4 domain of Vac8 was swapped for that of Meh1, another palmitoylated and N-myristoylated protein in yeast. In this case we saw specificity mimic that of wild type Vac8. Competition experiments revealed that the 11th armadillo repeat of Vac8 is an important element for recognition by Pfa3. This demonstrates that regions distant from the palmitoylated cysteines are important for recognition by DHHC proteins.S-Palmitoylation (hereafter referred to as palmitoylation) is a widespread post-translational modification in which the fatty acid palmitate (16:0) is attached to a cysteine residue via a thioester linkage. Palmitate can be released from a cysteine by hydrolysis of the thioester linkage; thus palmitoylation is a reversible modification. Numerous eukaryotic proteins are palmitoylated, and substrates include both peripheral and transmembrane domain proteins. The functional consequences of palmitoylation are diverse. Membrane association, protein stability, and protein trafficking are among the properties that can be modulated by the palmitoylation status of a protein (reviewed in 1, 2).The enzymes responsible for palmitoylation have only recently been identified. The first protein acyltransferases (PATs) 3 discovered were Erf2 and Akr1 in yeast, which palmitoylate Ras2 and Yck2, respectively (3, 4). Both Erf2 and Akr1 are transmembrane proteins with an Asp-His-(His/Tyr)-Cys (DHHC) motif embedded in a cysteine-rich domain (CRD). Homology with this domain has identified five additional DHHC proteins in Saccharomyces cerevisiae. These seven proteins account for the bulk of palmitoylating activity in the cell (5).To date 23 DHHC proteins have been identified in mammals (6). As the field has progressed DHHC proteins have been linked to the regulation of neurotransmission (7-9), nitric oxide release (10), and cell-cell contacts (11). Palmitoylation of Huntingtin by HIP14 (DHHC17) plays a protective role in ...

show abstract

Section: Vac8 Is Palmitoylated By Pfa3 At All Three N-terminal Cysteimentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Nadolski¹,

Linder

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…55 Vac8p may play a role in recruiting components of the homotypic fusion machinery into these microdomains. 58 It is worth noting that Tsc13p and Osh1p are involved in the biochemistry of the main components of lipid rafts in yeast, namely VLCFAs (constituents of sphingolipids) and ergosterol. 59 In conclusion, the complex role Vac8p plays in the mechanism of homotypic vacuole fusion warrants a closer look at its function in PMN.…”

Section: Lipid Homeostasis Is Important For the Biogenesis Of Pmn Vesmentioning

confidence: 99%

Nucleus-Vacuole Junctions and Piecemeal Microautophagy of the Nucleus inS. cerevisiae

Kvam

Goldfarb²

2007

Autophagy

111

117

View full text Add to dashboard Cite

Previously published online as an AcKnowlEDGEmEntSWe wish to thank past and present lab members who have contributed their ideas and technical assistance to this project, especially Jonathan Millen who made comments on this manuscript, and to our many helpful and supportive colleagues in the autophagy research community. This study was supported by grants from the National Science Foundation and National Institutes of Health. AbStrActVarious modes of autophagy conspire to degrade virtually every compartment of the eukaryotic cell. In Saccharomyces cerevisiae, a process called "piecemeal microautophagy of the nucleus" (PMN) even pinches off and degrades nonessential portions of the nucleus. PMN is a constitutive process induced to high levels by starvation or rapamycin, an inhibitor of TOR kinase. PMN occurs at nucleus-vacuole (NV) junctions, which are Velcro-like patches formed by interactions between the vacuole membrane protein Vac8p and the outer-nuclear-membrane protein Nvj1p. In response to nutrient depletion, Nvj1p increasingly binds and sequesters two proteins with roles in lipid metabolism, Osh1p and Tsc13p. Tsc13p is required for the normal biogenesis of PMN vesicles. The sequestration of Osh1p by Nvj1p likely serves to negatively regulate the trafficking of tryptophan permease(s) to the plasma membrane. Thus, NV junctions and PMN orchestrate novel and sophisticated responses to nutrient limitation.

show abstract

“…This hypotonic shock has previously been shown to induce vacuole fusion in vivo (37). Images were acquired using a Axioskop2 with a 100ϫ/1.4 NA plan apochromat oil immersion len (Zeiss), a CoolSnap HQ camera (Photometrics) and ImagePro Plus software (Media Cybernetics).…”

Section: Immunoprecipitation Of Actin-vacuole Protein Complexes and Dmentioning

confidence: 99%

Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling

Isgandarova

Jones

Forsberg

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.

show abstract

Fusion of Docked Membranes Requires the Armadillo Repeat Protein Vac8p

Cited by 83 publications

References 57 publications

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Nucleus-Vacuole Junctions and Piecemeal Microautophagy of the Nucleus inS. cerevisiae

Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling

Contact Info

Product

Resources

About