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Cited by 425Cellular transport and secretionFungal and yeast genetics researchAutophagy in Disease and Therapy

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New Component of the Vacuolar Class C-Vps Complex Couples Nucleotide Exchange on the Ypt7 Gtpase to Snare-Dependent Docking and Fusion

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Wurmser, Andrew E., Sato, Trey K., Emr, Scott D.·2000·The Journal of Cell Biology
DOI 10.1083/jcb.151.3.551PMC PMC2185595
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Abstract

The class C subset of vacuolar protein sorting (Vps) proteins (Vps11, Vps18, Vps16 and Vps33) assembles into a vacuole/prevacuole-associated complex. Here we demonstrate that the class C-Vps complex contains two additional proteins, Vps39 and Vps41. The COOH-terminal 148 amino acids of Vps39 direct its association with the class C-Vps complex by binding to Vps11. A previous study has shown that a large protein complex containing Vps39 and Vps41 functions as a downstream effector of the active, GTP-bound form of Ypt7, a rab GTPase required for the fusion of vesicular intermediates with the vacuole (Price, A., D. Seals, W. Wickner, and C. Ungermann. 2000. J. Cell Biol. 148:1231–1238). Here we present data that indicate that this complex also functions to stimulate nucleotide exchange on Ypt7. We show that Vps39 directly binds the GDP-bound and nucleotide-free forms of Ypt7 and that purified Vps39 stimulates nucleotide exchange on Ypt7. We propose that the class C-Vps complex both promotes Vps39-dependent nucleotide exchange on Ypt7 and, based on the work of Price et al., acts as a Ypt7 effector that tethers transport vesicles to the vacuole. Thus, the class C-Vps complex directs multiple reactions during the docking and fusion of vesicles with the vacuole, each of which contributes to the overall specificity and efficiency of this transport process.

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Figure 1 Multiple protein motifs exist within Vps39. (A) A BLAST search of the nonredundant protein database revealed homologues of Vps39 from H. sapiens, S. pombe, and C. elegans. (B) A multiple alignment (Clustal) of full-length Vps39 with these ORFs indicated that the COOH-terminus of Vps39 (amino acids 596–1,021) is 28, 25, and 22% identical and 45, 40, and 38% similar to the COOH terminal regions of the H. sapiens, S. pombe, and C. elegans ORFs, respectively. The conserved region of Vps39 contains two regions adjacent to the CHCR which we designate domain I and domain II.
Figure 1 Multiple protein motifs exist within Vps39. (A) A BLAST search of the nonredundant protein database revealed homologues of Vps39 from H. sapiens, S. pombe, and C. elegans. (B) A multiple alignment (Clustal) of full-length Vps39 with these ORFs indicated that the COOH-terminus of Vps39 (amino acids 596–1,021) is 28, 25, and 22% identical and 45, 40, and 38% similar to the COOH terminal regions of the H. sapiens, S. pombe, and C. elegans ORFs, respectively. The conserved region of Vps39 contains two regions adjacent to the CHCR which we designate domain I and domain II.CC BY-NC-SA · PubMed Central
Figure 2 Domain I and domain II are essential for Vps39 function. vps39Δ cells expressing the indicated single-copy protein A–VPS39 construct were labeled for 10 min with [35S]methionine and chased with excess unlabeled methionine for 30 min at 30°C. (A) Labeled lysates of these cells were immunoprecipitated for CPY or ALP and the resulting immunoprecipitates were analyzed by SDS-PAGE and fluorography. m, Mature; pro, precursor ALP. (B) Subcellular Vps39 localization was determined by osmotically lysing [35S]methionine-labeled spheroplasts and sequentially centrifuging the lysates at 13,000 g and 100,000 g to yield the low-speed pellet (P13), high-speed pellet (P100), and supernatent (S100). Vps39 was immunoprecipitated from P13, S100, and P100 fractions and analyzed by SDS-PAGE and fluorography. WT, Wild-type.
Figure 2 Domain I and domain II are essential for Vps39 function. vps39Δ cells expressing the indicated single-copy protein A–VPS39 construct were labeled for 10 min with [35S]methionine and chased with excess unlabeled methionine for 30 min at 30°C. (A) Labeled lysates of these cells were immunoprecipitated for CPY or ALP and the resulting immunoprecipitates were analyzed by SDS-PAGE and fluorography. m, Mature; pro, precursor ALP. (B) Subcellular Vps39 localization was determined by osmotically lysing [35S]methionine-labeled spheroplasts and sequentially centrifuging the lysates at 13,000 g and 100,000 g to yield the low-speed pellet (P13), high-speed pellet (P100), and supernatent (S100). Vps39 was immunoprecipitated from P13, S100, and P100 fractions and analyzed by SDS-PAGE and fluorography. WT, Wild-type.CC BY-NC-SA · PubMed Central
Figure 3 Vps39 and Vps41 are components of the C-Vps complex. Spheroplasts expressing single-copy protein (prot) A or protein A–VPS39 were labeled with [35S]methionine for 20 min and chased for 1 h at 30°C. Osmotic lysates of these strains were centrifuged at 100,000 g to generate the P total. 0.5% NP-40/250 mM NaCl extracts of the P total were incubated with IgG-sepharose to affinity-purify protein A fusions. Washed IgG-sepharose was analyzed by SDS-PAGE/fluorography. Multiple proteins coprecipitated preferentially with protein A–Vps39 (several are indicated by asterisks), but not protein A alone (lanes 1 and 2). IgG-sepharose–purified protein A fusions were also subjected to denaturation and immunoprecipitation using antibodies specific for Vps11, Vps18, Vps16, Vps33, and Vps41 (lanes 3–12).
Figure 3 Vps39 and Vps41 are components of the C-Vps complex. Spheroplasts expressing single-copy protein (prot) A or protein A–VPS39 were labeled with [35S]methionine for 20 min and chased for 1 h at 30°C. Osmotic lysates of these strains were centrifuged at 100,000 g to generate the P total. 0.5% NP-40/250 mM NaCl extracts of the P total were incubated with IgG-sepharose to affinity-purify protein A fusions. Washed IgG-sepharose was analyzed by SDS-PAGE/fluorography. Multiple proteins coprecipitated preferentially with protein A–Vps39 (several are indicated by asterisks), but not protein A alone (lanes 1 and 2). IgG-sepharose–purified protein A fusions were also subjected to denaturation and immunoprecipitation using antibodies specific for Vps11, Vps18, Vps16, Vps33, and Vps41 (lanes 3–12).CC BY-NC-SA · PubMed Central
Image 4
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Image 5
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Figure 5 ypt7 functionally interacts with vps39. Wild-type (WT), vps39tsf, or ypt7tsf strains were grown overnight at 26°C and pulse-labeled with [35S]methionine for 10 min at 26°C or 38°C. (A) After a 30-min chase, labeled wild-type, vps39tsf, or ypt7tsf spheroplasts were separated into intracellular (I) and extracellular (E) fractions. CPY immunoprecipitates of I and E were resolved by SDS-PAGE and fluorography, revealing CPY maturation defects in the vps39tsf and ypt7tsf at 38°C. API immunoprecipitates derived from labeled wild-type, vps39tsf, and ypt7tsf cells were analyzed by SDS-PAGE and fluorography, revealing defects in API maturation in the ypt7tsf, but not wild-type cells at 38°C. (B) CPY immunoprecipitates derived from the indicated strains were analyzed by SDS-PAGE and fluorography, revealing a strong genetic interaction with vps39tsf/ypt7tsf but not other double mutants at 26°C. m, Mature; p2, precursor CPY; pr, precursor API.
Figure 5 ypt7 functionally interacts with vps39. Wild-type (WT), vps39tsf, or ypt7tsf strains were grown overnight at 26°C and pulse-labeled with [35S]methionine for 10 min at 26°C or 38°C. (A) After a 30-min chase, labeled wild-type, vps39tsf, or ypt7tsf spheroplasts were separated into intracellular (I) and extracellular (E) fractions. CPY immunoprecipitates of I and E were resolved by SDS-PAGE and fluorography, revealing CPY maturation defects in the vps39tsf and ypt7tsf at 38°C. API immunoprecipitates derived from labeled wild-type, vps39tsf, and ypt7tsf cells were analyzed by SDS-PAGE and fluorography, revealing defects in API maturation in the ypt7tsf, but not wild-type cells at 38°C. (B) CPY immunoprecipitates derived from the indicated strains were analyzed by SDS-PAGE and fluorography, revealing a strong genetic interaction with vps39tsf/ypt7tsf but not other double mutants at 26°C. m, Mature; p2, precursor CPY; pr, precursor API.CC BY-NC-SA · PubMed Central
Figure 6 Vps39 physically associates with Ypt7. (A) Two-hybrid interactions between Vps39 and Ypt7 were assayed by quantifying the β-galactosidase activity of bait YPT7 and prey VPS39 cotransformants. The average β-galactosidase activity of three independent transformants of each strain is presented. (B) Two-hybrid interactions between Ypt7 and Vps39 require domain I. Association of the indicated Vps39 mutants with Ypt7 was assayed by cell growth under histidine-free conditions (>50 transformants tested). WT, wild-type.
Figure 6 Vps39 physically associates with Ypt7. (A) Two-hybrid interactions between Vps39 and Ypt7 were assayed by quantifying the β-galactosidase activity of bait YPT7 and prey VPS39 cotransformants. The average β-galactosidase activity of three independent transformants of each strain is presented. (B) Two-hybrid interactions between Ypt7 and Vps39 require domain I. Association of the indicated Vps39 mutants with Ypt7 was assayed by cell growth under histidine-free conditions (>50 transformants tested). WT, wild-type.CC BY-NC-SA · PubMed Central
Figure 7 Vps39 is a Ypt7 nucleotide exchange factor. (A) Purified protein A–Vps39 interacts with GDP-bound Ypt7. Protein A or protein A–Vps39 expressed from multicopy plasmids was purified for vps11Δ cells using IgG-sepharose. 50 ng of purified Ypt7 or Vps21 bound to GDP or GTPγS was incubated with IgG-bound protein A (lanes 3, 4, 7, and 8) or protein A–Vps39 (lanes 1, 2, 5, and 6) for 30 min at 25°C, washed multiple times, and the presence of Ypt7 (lanes 1–4) or Vps21 (lanes 5–8) was ascertained by Western blot. (B) The two-hybrid assay revealed preferential interactions between nucleotide-free Ypt7 and Vps39. β-Galactosidase activity was determined in cells cotransformed with prey VPS39 and bait constructs encoding mutants of YPT7 which bias Ypt7 to a specific nucleotide binding state or impair Ypt7–effector interactions. The average β-galactosidase activity of three independent transformants of each strain is presented. (C) 1 μM purified, soluble Ypt7 or Vps21 was prebound to unlabeled GDP, incubated with [3H]GTP (12 μM), and ∼20 ng of purified protein A or protein A–Vps39 for 5-, 15- or 25-min periods. Ypt7 or Vps21 was immobilized on nitrocellulose, washed, and the [3H]GTP exchanged onto Ypt7 or Vps21 was determined. A silver-stained gel of IgG-sepharose–purified protein A and protein A–Vps39 derived from 15 OD600 units of yeast is shown. IgG heavy chain, as well as a protein which comigrates with a degradation product of protein A–Vps39 (asterisk), is indicated.
Figure 7 Vps39 is a Ypt7 nucleotide exchange factor. (A) Purified protein A–Vps39 interacts with GDP-bound Ypt7. Protein A or protein A–Vps39 expressed from multicopy plasmids was purified for vps11Δ cells using IgG-sepharose. 50 ng of purified Ypt7 or Vps21 bound to GDP or GTPγS was incubated with IgG-bound protein A (lanes 3, 4, 7, and 8) or protein A–Vps39 (lanes 1, 2, 5, and 6) for 30 min at 25°C, washed multiple times, and the presence of Ypt7 (lanes 1–4) or Vps21 (lanes 5–8) was ascertained by Western blot. (B) The two-hybrid assay revealed preferential interactions between nucleotide-free Ypt7 and Vps39. β-Galactosidase activity was determined in cells cotransformed with prey VPS39 and bait constructs encoding mutants of YPT7 which bias Ypt7 to a specific nucleotide binding state or impair Ypt7–effector interactions. The average β-galactosidase activity of three independent transformants of each strain is presented. (C) 1 μM purified, soluble Ypt7 or Vps21 was prebound to unlabeled GDP, incubated with [3H]GTP (12 μM), and ∼20 ng of purified protein A or protein A–Vps39 for 5-, 15- or 25-min periods. Ypt7 or Vps21 was immobilized on nitrocellulose, washed, and the [3H]GTP exchanged onto Ypt7 or Vps21 was determined. A silver-stained gel of IgG-sepharose–purified protein A and protein A–Vps39 derived from 15 OD600 units of yeast is shown. IgG heavy chain, as well as a protein which comigrates with a degradation product of protein A–Vps39 (asterisk), is indicated.CC BY-NC-SA · PubMed Central
Figure 8 Proposed functional roles of the C-Vps complex (Vps39, Vps41, Vps11, Vps18, Vps16, Vps33) in the fusion of prevacuolar hydrolase-containing cargo vesicles with the vacuole. Our observations support a model in which association of Vps39 with the C-Vps complex localizes Vps39 to Ypt7 on cargo vesicles and the vacuole, enabling Vps39 to convert inactive Ypt7GDP to its active GTP-bound state (step 1). Vam3 (t-SNARE) is maintained in an unpaired state through interactions with the C-Vps complex. During step 2, Ypt7GTP associates with an effector protein(s) contained within the C-Vps complex, tethering these compartments such that Vti1 (v-SNARE) and Vam3 are brought into close proximity. At step 3, the C-Vps complex releases Vam3, allowing trans-SNARE complexes containing Vti1, Vam3, and Vam7 (SNAP25) to form and drive membrane fusion.
Figure 8 Proposed functional roles of the C-Vps complex (Vps39, Vps41, Vps11, Vps18, Vps16, Vps33) in the fusion of prevacuolar hydrolase-containing cargo vesicles with the vacuole. Our observations support a model in which association of Vps39 with the C-Vps complex localizes Vps39 to Ypt7 on cargo vesicles and the vacuole, enabling Vps39 to convert inactive Ypt7GDP to its active GTP-bound state (step 1). Vam3 (t-SNARE) is maintained in an unpaired state through interactions with the C-Vps complex. During step 2, Ypt7GTP associates with an effector protein(s) contained within the C-Vps complex, tethering these compartments such that Vti1 (v-SNARE) and Vam3 are brought into close proximity. At step 3, the C-Vps complex releases Vam3, allowing trans-SNARE complexes containing Vti1, Vam3, and Vam7 (SNAP25) to form and drive membrane fusion.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Wurmser, A. E., Sato, T. K., & Emr, S. D.
Affiliations
Howard Hughes Medical Institute (US)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2000-10-30
Volume
151
Issue
3
Pages
551-562
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.151.3.551
PMC
PMC2185595
OpenAlex
W2132005005
Cited by
425
References
48
Citations by year
2026: 4 · 2025: 8 · 2024: 10 · 2023: 12 · 2022: 7 · 2021: 12 · 2020: 10 · 2019: 13 · 2018: 8 · 2017: 16 · 2016: 16 · 2015: 15 · 2014: 23 · 2013: 17 · 2012: 28
FWCI
8.09
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5p01ca058689-10)
  • National Cancer Institute (CA 58689)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Keywords
VPS39, VPS41, Ypt7, Rab GTPases, nucleotide exchange, vacuole fusion, vesicle docking, SNARE proteins, vacuolar protein sorting, yeast vacuole, protein complex formation
MeSH terms
Amino Acid Sequence, Biological Transport, Carrier Proteins, Fungal Proteins, Guanosine Triphosphate, Membrane Fusion, Membrane Proteins, Models, Biological, Molecular Sequence Data, Mutation, Nuclear Proteins, Protein Binding, Saccharomyces cerevisiae, Substrate Specificity, Vacuoles, Sequence Alignment, RNA-Binding Proteins, Conserved Sequence, Protein Structure, Tertiary, Genes, Essential, rab GTP-Binding Proteins, Two-Hybrid System Techniques, Saccharomyces cerevisiae Proteins, Vesicular Transport Proteins, Adaptor Proteins, Vesicular Transport, Macromolecular Substances, SNARE Proteins

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