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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 Tlg2p in isolation from its SNARE binding partners. (A) Tlg1p- and (B) Tlg2p-containing complexes were immunoprecipitated from the wild-type (WT) (NOzY21) or vps21Δ (NOzY24) cells. Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes as indicated.
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fig2: Vps45p binds to Tlg2p in isolation from its SNARE binding partners. (A) Tlg1p- and (B) Tlg2p-containing complexes were immunoprecipitated from the wild-type (WT) (NOzY21) or vps21Δ (NOzY24) cells. Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes as indicated.

Mentions: We, and others, have shown that Vps45p binds to monomeric Tlg2p in vitro (Bryant and James, 2001; Dulubova et al., 2002; Yamaguchi et al., 2002). The observation that Sec1p binds to Ssop-containing SNARE complexes, but not to the monomeric t-SNARE (Carr et al., 1999), raises the possibility that the association observed between Tlg2p and Vps45p in vivo does not reflect the SM protein binding to the monomeric t-SNARE, but rather to the cis-SNARE complex. This is the case for the interaction between Sec1p and Ssop (Carr et al., 1999), as demonstrated by the lack of interaction between these two proteins in sec4–8 cells (Carr et al., 1999). SEC4 encodes the Rab protein involved in the fusion of secretory vesicles with the plasma membrane, and sec4–8 cells are defective in assembly of Ssop SNARE complexes (Grote and Novick, 1999). The Rab protein involved in VPS45-dependent membrane transport is encoded by VPS21 (Gerrard et al., 2000). Fig. 2 shows that Tlg1p was not found in complex with its SNARE binding partners in vps21Δ mutant cells. Under these conditions, Tlg1p no longer coprecipitated Vps45p (Fig. 2 A). Importantly, Vps45p was still found in complex with Tlg2p in vps21Δ cells (Fig. 2 B). These data demonstrate that Vps45p binds to the monomeric t-SNARE Tlg2p, and to the cis-Tlg2p–Tlg1p–Vti1p–Snc2p SNARE complex in vivo.


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 Tlg2p in isolation from its SNARE binding partners. (A) Tlg1p- and (B) Tlg2p-containing complexes were immunoprecipitated from the wild-type (WT) (NOzY21) or vps21Δ (NOzY24) cells. Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes as indicated.
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Related In: Results  -  Collection

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fig2: Vps45p binds to Tlg2p in isolation from its SNARE binding partners. (A) Tlg1p- and (B) Tlg2p-containing complexes were immunoprecipitated from the wild-type (WT) (NOzY21) or vps21Δ (NOzY24) cells. Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes as indicated.
Mentions: We, and others, have shown that Vps45p binds to monomeric Tlg2p in vitro (Bryant and James, 2001; Dulubova et al., 2002; Yamaguchi et al., 2002). The observation that Sec1p binds to Ssop-containing SNARE complexes, but not to the monomeric t-SNARE (Carr et al., 1999), raises the possibility that the association observed between Tlg2p and Vps45p in vivo does not reflect the SM protein binding to the monomeric t-SNARE, but rather to the cis-SNARE complex. This is the case for the interaction between Sec1p and Ssop (Carr et al., 1999), as demonstrated by the lack of interaction between these two proteins in sec4–8 cells (Carr et al., 1999). SEC4 encodes the Rab protein involved in the fusion of secretory vesicles with the plasma membrane, and sec4–8 cells are defective in assembly of Ssop SNARE complexes (Grote and Novick, 1999). The Rab protein involved in VPS45-dependent membrane transport is encoded by VPS21 (Gerrard et al., 2000). Fig. 2 shows that Tlg1p was not found in complex with its SNARE binding partners in vps21Δ mutant cells. Under these conditions, Tlg1p no longer coprecipitated Vps45p (Fig. 2 A). Importantly, Vps45p was still found in complex with Tlg2p in vps21Δ cells (Fig. 2 B). These data demonstrate that Vps45p binds to the monomeric t-SNARE Tlg2p, and to the cis-Tlg2p–Tlg1p–Vti1p–Snc2p SNARE complex in vivo.

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