Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation

Abstract

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.

Fig. 1. Vps45p interacts with Tlg2p. (A) Vps45p is membrane localized through Tlg2p. Wild-type (SF838-9D), pep12Δ (SGY39) and tlg2Δ (NOzY3) cells were fractionated by differential centrifugation to yield a low-speed pellet (P13), a high-speed pellet (P100) and a soluble fraction (S100). The amount of Vps45p, Pep12p, Tlg2p and Pgk1p in each fraction was assessed using immunoblot analysis. (B) Vps45p binds to the cytosolic domain of Tlg2p. Glutathione–Sepharose beads loaded with GST–Pep12p, GST–Sed5p, GST–Tlg2p, GST–Tlg2-Δ1–230, GST alone or empty beads (0) were incubated with [³⁵S]methionine-labelled, in vitro-translated, Vps45p. GST fusions and associated proteins were eluted and analysed using SDS–PAGE and fluorography.

Fig. 2. Tlg2p is down-regulated upon loss of Vps45p. Proteins contained within whole-cell extracts prepared from wild-type (RPY10), vps45Δ (NOzY2) and tlg2Δ (NOzY4) cells were separated using SDS–PAGE before being transferred to nitrocellulose. The resulting filters were probed using antibodies that specifically recognize Tlg2p, Vti1p, Pep12p, Vam3p, Ufe1p, Sed5p or Tlg1p as indicated.

Fig. 3. Loss of VPS45 results in degradation of Tlg2p by the proteasome. (A) Proteins contained within whole-cell extracts prepared from wild-type (RPY10), vps45Δ (NOzY2), pep4-3 (SF838–9D), pep4-3 vps45Δ (NOzY1), pre1-1 pre2-2 (LHY58), pre1-1 pre2-2 vps45Δ (NOzY13), cim3-1 (LHY913) and cim3-1 vps45Δ (NOzY8) were separated using SDS–PAGE before being transferred to nitrocellulose. The resulting filter was probed using antibodies that specifically recognize Tlg2p. (B) Proteins synthesized by wild-type (RPY10), vps45Δ (NOzY2), vps45Δ pep4-3 (NOzY1) and vps45Δ pre1-1 pre2-2 (NOzY13) cells were labelled using [³⁵S]cysteine/methionine for 10 min prior to the addition of excess unlabelled cysteine and methionine. At the times indicated, Tlg2p was immunoprecipitated and samples were analysed using SDS–PAGE and fluorography.

Fig. 4. Vps45p is required for Tlg2p to form ternary complexes with Tlg1p and Vti1p. (A) Stabilized Tlg2p does not co-precipitate Tlg1p and Vti1p. Tlg2p-containing complexes were immunoprecipitated from the following strains: wild-type (LHY55), tlg2Δ (NozY4), vps45Δ (NozY11), pre1-1 pre2-2 (LHY58) and vps45Δ pre1-1 pre2-2 (NOzY13). Immunoblot analysis was used to detect the presence of Tlg1p and Vti1p in the immuno precipitated complexes. As a control, proteins precipitated using pre-immune serum from wild-type and tlg2Δ cells (PI) were also analysed. (B) Tlg2p is no longer found in SNARE complexes following rapid inactivation of Vps45p. Tlg2p-containing complexes were immunoprecipitated from wild-type (SF838-9D) and vps45-ts (RPY15) cells. Cells were grown to early log phase at 25°C before being shifted to 37°C for the indicated times. Immunoblot analysis was used to detect the presence of Tlg1p and Vti1p in the immunoprecipitated complexes.

Fig. 5. Tlg2p is targeted independently of Vps45p. Total membranes from wild-type (LHY55) and vps45Δ pre1-1 pre2-2 (NOzY13) cells were fractionated to equilibrium on a 40–65% self-forming sorbitol gradient. The amount of Tlg2p, Vph1p, Dpm1p and Pep12p in each fraction was assessed through immunoblot analysis.

Fig. 6. Truncation of Tlg2p by-passes the requirement for SNARE complex formation on VPS45. (A) Tlg2p-Δ1–230 does not recruit Vps45p to membranes. tlg2Δ cells (NozY3) expressing either full-length Tlg2p (from pNOz7) or Tlg2p-Δ1–230 (from pNOz8) were fractionated using differential centrifugation as in Figure 1. The amount of Vps45p contained within each fraction was determined by immunoblot analysis. (B) Tlg2p-Δ1–230 binds Tlg1p and Vti1p in the absence of VPS45. vps45Δ pre1-1 pre2-2 (NOzY13) cells harbouring a plasmid encoding either Tlg2p-Δ1–230 (pNOz8) or full-length Tlg2p (pNOz7) under control of the GAL1/10 promoter were grown using either glucose (SD) or galactose (Sgal/Raff) as a carbon source. Antibodies that recognize both the full-length Tlg2p and the truncated Tlg2p-Δ1–230 were used to precipitate Tlg2p-containing complexes from lysates of these cells. The amount of Tlg1p and Vti1p in these complexes was assessed through immunoblot analysis. (C) Tlg2p-Δ1–230 partially overcomes CPY missorting in vps45Δ cells. Proteins synthesized by wild-type cells (RPY10), vps45Δ cells (NozY2) and vps45Δ cells producing Tlg2p-Δ1–230 (NOzY2 transformed with pNOz8) were grown using galactose as a carbon source (Sgal/Raff) and metabolically labelled for a 10 min pulse using [³⁵S]methionine. A chase period of 30 min was initiated by the addition of excess unlabelled methionine, after which time CPY was immunoprecipitated from both intracellular (I) and extracellular (E) fractions.