Proteins was dialyzed into PS buffer containing 125 mM KCl and 5 mM MgCl2, frozen in aliquots with water nitrogen, and found in fusion reactions in 1 g/ml

Proteins was dialyzed into PS buffer containing 125 mM KCl and 5 mM MgCl2, frozen in aliquots with water nitrogen, and found in fusion reactions in 1 g/ml. Immunoprecipitations Sedimented vacuoles had been overlaid with ice-cold solubilization buffer (20 mM HEPES/KOH, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100 (Anatrace), 20% glycerol, Copper PeptideGHK-Cu GHK-Copper 1 protease inhibitor cocktail (0.46 g/ml leupeptin, 3.5 g/ml pepstatin, 2.4 g/ml pefabloc-SC, 1 mM PMSF)), resuspended on ice, and diluted to 0.15C0.3 mg vacuole proteins/ml. through GTP-dependent organizations with effectors prior to (Rothman and Stevens, NS-018 maleate 1986), (Jones, 1977), and (Wada et al, 1992) phenotypes of defective vacuole trafficking, function, and structure. Homologous genes in other organisms regulate pigment transport to lysosomes and storage granule compartments (Warner et al, 1998; Sevrioukov et al, 1999; Suzuki et al, 2003). Distinct phenotypes arise upon deletion of the genes encoding yeast HOPS subunits. Class B mutants (Vps39 and 41) have moderate vacuole fragmentation, while Class C (Vps11, 16, 18, and 33) mutants cause severe vacuole fragmentation. The complex of the four Class C Vps proteins associates with accessory factors such as either Vps39p/Vps41p or Vps8p to mediate distinct trafficking and fusion events (Sato et al, 2000; Seals et al, 2000; Richardson et al, 2004; Subramanian et al, 2004). HOPS can stimulate NS-018 maleate nucleotide exchange on Ypt7p and specifically associates with GTP-bound Ypt7p (Seals et al, 2000; Wurmser et al, 2000). Since one HOPS subunit, Vps33p, is an SM family member, Ypt7p interactions may regulate HOPS associations with vacuole SNAREs. The integration of these functions in a single protein complex makes HOPS a unique model to study the intersection between Rab and SNARE functions. Yeast vacuole fusion, reconstituted (Mayer and Wickner, 1997; Ungermann et al, 1998b; Wang et al, 2002). SNARE complex formation leads to calcium release from lumenal stores, bilayer fusion, and aqueous compartment mixing (Peters and Mayer, 1998; Weber et al, 1998; Merz and Wickner, 2004b). To explore the interactions between SNAREs and their chaperones, we have isolated vacuolar SNARE-associated complexes. Although we have previously shown that HOPS associates with Vam3p and Nyv1p (Price et al, 2000a), only a small fraction of HOPS or SNAREs associates in this manner. We now report that vacuole SNAREs are found in four distinct pools. The first is dissociated SNAREs. The remaining SNARE complexes are associated with Sec17p, HOPS, or neither. These SNARE complex associations have distinct functions during vacuole fusion. Sec17p-containing SNARE complexes are disassembled by Sec18p to establish a pool of unpaired SNAREs, which are required for subsequent interactions in and function normally for vacuole fusion (Wang et al, 2002, 2003b). We NS-018 maleate now use these GFP tags to study the physical associations of SNAREs. Vacuoles purified from strains expressing GFP-tagged proteins or from their untagged parental strains were solubilized with Triton X-100 and immunoprecipitated with antibodies to GFP. Immunoisolated complexes of three GFP-tagged SNAREs (Vam7p, Vti1p, and Vam3p) were subjected to SDSCPAGE and silver staining (Figure 1, top). A total of 11 bands were found specifically in immunoprecipitates from tagged Vam7p and Vam3p strains (10 for Vti1p). Other specifically associated polypeptides were breakdown products of tagged SNAREs. Open in a separate window Figure 1 Isolation of SNARE complexes from yeast vacuoles. Vacuoles isolated from (A) untagged (DKY6281) and Vam7-GFP2, (B) untagged (BJ2168) and Vti1-GFP, and (C) untagged (BJ3505) and GFP-Vam3p-tagged yeast strains were solubilized and immunoprecipitated with antibodies to GFP (see Materials and methods). Immunoprecipitates were analyzed by SDSCPAGE, silver staining (top), and immunoblotting (bottom). Vacuoles were prepared by large-scale fermentation and batch purification (Ungermann et al, 1999; Seals et al, 2000). Vacuoles in PS buffer (120 g) were reisolated by centrifugation (11 000 vacuole fusion (Thorngren et al, 2004). This provides an opportunity to examine the role of HOPS during SNARE complex assembly and vacuole fusion in the absence of Sec17/18p-mediated disassembly. Fusion reactions can be driven by added Vam7p in the continued presence of anti-Sec17p, which traps any free Sec17p and prevents SNARE complex disassembly (Boeddinghaus et al, 2002). We used these conditions and a GST-Vam7p fusion protein to study the associations of SNARE proteins from the unpaired state prior to vacuole fusion NS-018 maleate (Thorngren et al, 2004). Fusion reactions were performed in the presence of ATP and antibodies to Sec17p. Added GST-Vam7p NS-018 maleate bypassed the anti-Sec17p block and allowed fusion (Figure 5B and C). The added GST-Vam7p formed a complex with Vam3p, Vti1p, Nyv1p, and HOPS, but not with untagged Vam7p (Figure 5D, lane 8). SNARE complex assembly and fusion was.