1. Class C Vps Protein Complex Regulates Vacuolar SNARE Pairing and Is Required for Vesicle Docking/Fusion 
Trey K. Sato, Peter Rehling, Michael R. Peterson, Scott D. Emr 
Molecular Cell 
Volume 6, Issue 3, Pages (September 2000)
DOI: /S (00)

2. Figure 1 Class C vps Mutants Genetically Interact with VAM3
(A) Analysis of vacuolar protein transport in the vps16tsf mutant. Strain BHY113 transformed with pMP86 (wild-type) or pMP100 (vps16tsf) were preincubated at 26°C or 38°C for 10 min prior to pulse labeling for 10 min with [35S]cysteine/methionine at the same temperature and chased for the indicated times. ALP and CPY were immunoprecipitated from cell lysates and resolved by SDS–PAGE.
(B) Genetic interactions between vam3tsf and late-acting vps mutants. Wild-type (TDY2 + pVAM3.414), vam3tsf (TDY2 + pVAM ), and indicated double mutant strains were pulse-labeled for 10 min and chased for 45 min at 26°C. ALP was immunoprecipitated from the indicated strains.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2 Vps18 and Vps33 Interact with Vam3
(A and B) Coimmunoprecipitation of Vps18-HA (A) and Vps33-HA (B). Equal amounts of isolated vacuoles from the indicated strains were subjected to immunoprecipitation with anti-HA antibodies against Vps18-HA ([A], lanes 5–8) and Vps33-HA ([B], lanes 5–8). Lanes 1–4 show the total of the assayed vacuolar proteins; 1 μg protein was loaded per lane. Twenty percent of the immunoprecipitated protein was loaded per lane on an SDS–PAGE gel, and bound protein was detected by Western blotting.
(C) In vitro binding analyses. The indicated GST fusion proteins were expressed and purified from E. coli using glutathione-Sepharose. Equal amounts of immobilized fusion proteins were incubated with detergent-solubilized extracts (25 mg of total protein) prepared from yeast strains expressing Vps18-HA or Vps33-HA. Following incubation, immobilized fusion proteins were washed and eluted with sample buffer. Twenty percent of the total eluate was loaded per lane; the amount of solubilized lysate that was loaded corresponds to approximately 5% of the total input. The top panel displays a Western blot with anti-HA antibody, while the bottom panel is the corresponding Coomassie-stained gel showing the input of immobilized GST fusion proteins.
(D) Coimmunoprecipitation of Vps33-HA from sec18-1 mutant cells. Equal amounts of isolated vacuoles from strain PRY18 (sec18-1 VPS18-HA) or TKSY91 (sec18-1 VPS33-HA) incubated at 26°C or 37°C for 30 min were solubilized in Triton X-100. Solubilized material was subjected to immunoprecipitation with anti-HA antibodies. Lanes 1 and 2 represent total protein from 1 μg of purified vacuoles. In lanes 3 and 4, 20% of the precipitated protein was loaded per lane on an SDS–PAGE gel and detected by Western blotting.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 Homotypic Vacuole Fusion Requires Vps18
In vitro fusion of vacuoles. Vacuoles from pep4Δ and pho8Δ strains that both expressed Vps18-HA (A) or vacuoles from pep4Δ and pho8Δ strains in which only one partner expressed Vps18-HA (B) were isolated and incubated with anti-HA antibodies and ATP in combinations indicated for standard fusion reactions. Maximum activity after the 90 min fusion reaction (100%) was 1.54 U. Both (A) and (B) show one representative experiment. Values shown are averages of duplicate measurements.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4 Association between Vps33 and Vam3 Is Dependent upon Vps18
(A) Isolation of the C-Vps complex. Two hundred micrograms of purified vacuoles from wild-type or vps16Δ strains expressing Protein A or Protein A-Vps16, respectively, were solubilized with Triton X-100. Associated proteins were isolated with IgG-coated Dynabeads. Forty percent of the eluted protein was resolved by SDS–PAGE and visualized by silver staining (bottom panel) or Western blotting with anti-Vam3 or Vam7 antibodies (upper panel). Lanes 1 and 2 represent the total protein from 1 μg of vacuoles. The asterisk (*) denotes a protein band not detected with Protein A alone.
(B) Identification of C-Vps complex components. Detergent-solubilized extracts from the indicated HA-tagged strains expressing Protein A or Protein A-Vps16 fusion protein were prepared as in Experimental Procedures. Proteins bound to Protein A or Protein A-Vps16 were isolated by IgG-affinity chromatography and visualized by Western blotting with anti-HA antibodies. Approximately 25% of the total eluate was loaded per lane.
(C) In vitro binding of Vps18-HA and Vps33-HA with Vam3 in mutant strains. GST or GST-Vam3 fusion protein immobilized to glutathione-Sepharose was incubated with detergent-extracts (20 mg total protein) prepared from mutant strains expressing Vps18-HA or Vps33-HA. After incubation and washing, bound protein was eluted with sample buffer. Approximately 20% of the total eluate was loaded per lane. The top panel is a Western blot of the eluted proteins with anti-HA antibody, while the bottom panel displays the GST-fusion protein input by Coomassie staining.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5 Protein A-Vam7 Does Not Associate with the C-Vps Complex
(A and B) IgG-affinity chromatography of Protein A-Vam7 fusion protein. Detergent-solubilized extracts from the indicated vam7Δ mutant strains expressing Protein A or Protein A-Vam7 fusion protein were prepared as described in Experimental Procedures. From these extracts, protein bound to Protein A or Protein A-Vam7 was isolated by IgG-affinity chromatography and visualized by Western blotting ([A], lanes 1–5). In (B), 150 μg of vacuoles from sec18-1 mutant cells expressing Protein A-Vam7 along with Vps18-HA or Vps33-HA incubated at 26°C or 37°C was subjected to IgG-affinity chromatography. Associated proteins were eluted and visualized by Western blotting. Approximately 20% of the eluate was loaded per lane.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6 C-Vps Complex Function Is Essential for SNARE Pairing
(A) Vacuolar SNARE pairing in Class C vps null mutants. Vam3 bound to Protein A-Vam7 was isolated by IgG-affinity chromatography from detergent-solubilized extracts produced from the indicated strains expressing the Protein A-Vam7 fusion protein and detected by Western blotting.
(B) Vacuolar SNARE pairing in vps18tsf mutant cells. vam7Δ or vps18tsf vam7Δ mutant cells expressing Protein A-Vam7 were incubated at 38°C for the indicated times. Associated proteins were isolated by IgG-affinity chromatography and visualized by Western blotting. Amounts of Vam3 bound to Protein A-Vam7 were quantified by densitometry. Values were normalized to the amount of Protein A-Vam7 detected at 0 min for each strain. The histogram displays the percentage of bound Vam3 compared to the amount of Vam3 bound at 0 min for each strain.
(C) Vacuolar SNARE pairing in other temperature-conditional vps mutants. Protein A-Vam7 fusion protein was expressed in the indicated temperature-conditional mutant strains. Strains were incubated at 26°C or 38°C for 30 min prior to harvesting. The association of Vam3 and Vti1 with Protein A-Vam7 was assayed by IgG-affinity chromatography and Western blotting.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
Model for the Function of the C-Vps Complex in Vacuolar Protein Transport
A schematic diagram of proteins that function in the docking/fusion of cargo vesicles to the vacuole. The stages of membrane fusion are in italics. Components of the C-Vps complex are represented by numbered geometric shapes (18, Vps18; 11, Vps11; 16, Vps16; 33, Vps33). SNAREs are represented by shaded rectangles (black, Vti1; light gray, Vam7; dark gray, Vam3). Tethering is mediated by Ypt7, Vam2/Vps41, and Vam6/Vps39.
Molecular Cell 2000 6, DOI: ( /S (00) )