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The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport.

Bryant NJ, James DE - J. Cell Biol. (2003)

Bottom Line: Here, we show that glc7 mutant cells accumulate SNARE complexes.These complexes are clearly different from those found in either wild-type or sec18-1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them.Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation.

View Article: PubMed Central - PubMed

Affiliation: The Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia 2010. n.bryant@bio.gla.ac.uk

ABSTRACT
Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion. During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer. Here, we show that glc7 mutant cells accumulate SNARE complexes. These complexes are clearly different from those found in either wild-type or sec18-1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them. Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes. Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation. These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle.

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Vps45p binds to cis-Tlg1p–Tlg2p–Vti1p–Snc2p SNARE complexes that accumulate upon inactivation of SEC18. Tlg1p-containing complexes were immunoprecipitated from the wild-type (WT) cells (NOzY21) or sec18–1 cells (NOzY22) grown at 25°C (25°C), or grown at 25°C, and then incubated at 37°C for 10 min before cell lysate preparation (37°C). Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes.
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fig1: Vps45p binds to cis-Tlg1p–Tlg2p–Vti1p–Snc2p SNARE complexes that accumulate upon inactivation of SEC18. Tlg1p-containing complexes were immunoprecipitated from the wild-type (WT) cells (NOzY21) or sec18–1 cells (NOzY22) grown at 25°C (25°C), or grown at 25°C, and then incubated at 37°C for 10 min before cell lysate preparation (37°C). Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes.

Mentions: We have demonstrated previously that Vps45p binds to Tlg2p both in vitro and in vivo (Bryant and James, 2001). Other SM proteins, including Sly1p and Sec1p, bind to their respective SNARE complexes (Kosodo et al., 1998; Carr et al., 1999; Peng and Gallwitz, 2002). Importantly, Sec1p binds to the cis-core complex, indicating a role for SM proteins after vesicle fusion (Grote et al., 2000). To determine whether Vps45p binds to the Tlg2p-containing core complex, we took advantage of cells harboring the temperature-sensitive sec18–1 mutation. Sec18p is an ATPase that catalyses the disassembly of SNARE complexes so that they can be recycled for further rounds of vesicle transport (Mayer et al., 1996). At the restrictive temperature (37°C), the mutant Sec18–1p is deficient in this ATPase activity and sec18–1 cells, thus, accumulate cis-SNARE complexes (Grote et al., 2000). Consistent with this, we observed a significant increase in the accumulation of cis-Tlg2p–Tlg1p–Vti1p–Snc2p SNARE complexes in sec18–1 cells placed at the nonpermissive temperature (37°C). Fig. 1 demonstrates that there is a five- to sevenfold increase in the amount of Tlg2p, Vti1p, and Snc2p that coprecipitate with Tlg1p under these conditions. Commensurate with the increase in SNARE complexes, we observed a fivefold increase in the amount of Vps45p that coprecipitated with Tlg1p. As we have previously demonstrated that Vps45p does not bind directly to Tlg1p (Bryant and James, 2001), these data indicate that Vps45p, like Sec1p (Carr et al., 1999) and Sly1p (Kosodo et al., 2002; Peng and Gallwitz, 2002), binds to its cognate SNARE complex. Given that, we observed the association of Vps45p with SNARE complexes in sec18–1 cells, this likely corresponds to an association with cis-complexes, and is in agreement with previous studies (Carr et al., 1999; Grote et al., 2000).


The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport.

Bryant NJ, James DE - J. Cell Biol. (2003)

Vps45p binds to cis-Tlg1p–Tlg2p–Vti1p–Snc2p SNARE complexes that accumulate upon inactivation of SEC18. Tlg1p-containing complexes were immunoprecipitated from the wild-type (WT) cells (NOzY21) or sec18–1 cells (NOzY22) grown at 25°C (25°C), or grown at 25°C, and then incubated at 37°C for 10 min before cell lysate preparation (37°C). Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199362&req=5

fig1: Vps45p binds to cis-Tlg1p–Tlg2p–Vti1p–Snc2p SNARE complexes that accumulate upon inactivation of SEC18. Tlg1p-containing complexes were immunoprecipitated from the wild-type (WT) cells (NOzY21) or sec18–1 cells (NOzY22) grown at 25°C (25°C), or grown at 25°C, and then incubated at 37°C for 10 min before cell lysate preparation (37°C). Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes.
Mentions: We have demonstrated previously that Vps45p binds to Tlg2p both in vitro and in vivo (Bryant and James, 2001). Other SM proteins, including Sly1p and Sec1p, bind to their respective SNARE complexes (Kosodo et al., 1998; Carr et al., 1999; Peng and Gallwitz, 2002). Importantly, Sec1p binds to the cis-core complex, indicating a role for SM proteins after vesicle fusion (Grote et al., 2000). To determine whether Vps45p binds to the Tlg2p-containing core complex, we took advantage of cells harboring the temperature-sensitive sec18–1 mutation. Sec18p is an ATPase that catalyses the disassembly of SNARE complexes so that they can be recycled for further rounds of vesicle transport (Mayer et al., 1996). At the restrictive temperature (37°C), the mutant Sec18–1p is deficient in this ATPase activity and sec18–1 cells, thus, accumulate cis-SNARE complexes (Grote et al., 2000). Consistent with this, we observed a significant increase in the accumulation of cis-Tlg2p–Tlg1p–Vti1p–Snc2p SNARE complexes in sec18–1 cells placed at the nonpermissive temperature (37°C). Fig. 1 demonstrates that there is a five- to sevenfold increase in the amount of Tlg2p, Vti1p, and Snc2p that coprecipitate with Tlg1p under these conditions. Commensurate with the increase in SNARE complexes, we observed a fivefold increase in the amount of Vps45p that coprecipitated with Tlg1p. As we have previously demonstrated that Vps45p does not bind directly to Tlg1p (Bryant and James, 2001), these data indicate that Vps45p, like Sec1p (Carr et al., 1999) and Sly1p (Kosodo et al., 2002; Peng and Gallwitz, 2002), binds to its cognate SNARE complex. Given that, we observed the association of Vps45p with SNARE complexes in sec18–1 cells, this likely corresponds to an association with cis-complexes, and is in agreement with previous studies (Carr et al., 1999; Grote et al., 2000).

Bottom Line: Here, we show that glc7 mutant cells accumulate SNARE complexes.These complexes are clearly different from those found in either wild-type or sec18-1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them.Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation.

View Article: PubMed Central - PubMed

Affiliation: The Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia 2010. n.bryant@bio.gla.ac.uk

ABSTRACT
Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion. During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer. Here, we show that glc7 mutant cells accumulate SNARE complexes. These complexes are clearly different from those found in either wild-type or sec18-1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them. Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes. Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation. These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle.

Show MeSH
Related in: MedlinePlus