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. 1999 Dec;10(12):4149-61.
doi: 10.1091/mbc.10.12.4149.

Promiscuity in Rab-SNARE interactions

E Grote  1 , P J Novick
Affiliations
Free PMC article

Promiscuity in Rab-SNARE interactions

E Grote et al. Mol Biol Cell. 1999 Dec.
Free PMC article

Abstract

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

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Figures

Figure 1
Figure 1
Snc2p binds to diverse t-SNAREs. (A) Ssop, Pep12p, Sed5p, and Vam3p coimmunoprecipitate with HA-Snc2p. Lysates were prepared from either wild-type yeast (NY605) or sec18-1 yeast expressing HA-Snc2p (NY1643). The cells were shifted to 37°C for 10 min before lysis to allow SNARE complexes to accumulate in the sec18-1 mutant strain. An immunoblot from anti-HA and no antibody control immunoprecipitates was probed for coprecipitating t-SNAREs. The minimal amount of nonspecific Ssop precipitation from the untagged strain is independent of Sec18p function. (B) myc-Sso2 does not bind to HA-Snc2p in vitro. HA-Snc2p and myc-Ssop were expressed in different populations of sec18-1 cells (NY1643 and NY1727), which were mixed, shifted to 37°C for 10 min, and then lysed. HA-Snc2 was immunoprecipitated with anti-HA antibodies, native and HA-tagged Sncp were immunoprecipitated with anti-Sncp antibodies, and a control precipitation was performed without antibody. The immunoprecipitates were probed for coprecipitation of myc-Sso2p and native Sso proteins with biotinylated anti-Sso antibodies. The anti-Sncp antibody reacts with an epitope conserved between Snc1p and Snc2p. Likewise, the anti-Ssop antibody reacts with an epitope conserved between Sso1p and Sso2p. (C) Pep12p does not bind to HA-Snc2p in vitro. Pep12p and HA-Snc2p were either coexpressed in the same SEC+ cells (NY1722) or expressed in different cells that were mixed before lysis (NY605and NY1726). Anti-HA immunoprecipitates were probed for coprecipitation of Pep12p. An immunoblot of the lysates was probed with antibodies against Sncp and Pep12p.
Figure 2
Figure 2
SNARE complex assembly depends on membrane transport. (A) Mutations in three different Rab proteins inhibit Sncp association with both Ssop and Tlg2. Wild-type (DBY1034), ypt1-A136D, ΔYPT31 ypt32-A141D, and sec4-8 cells were shifted to the restrictive temperature of 33°C for 10 min before homogenization. Anti-Ssop and anti-Tlg2p immunoprecipitates were probed for coprecipitation of Sncp. (B) Early secretion blocks inhibit Ssop association with Sncp. Wild-type (NY13) and sec mutant strains were grown to log phase at 25°C and then shifted to 37°C for 0, 10, or 30 min before homogenization. Anti-Sncp immunoprecipitates were probed for coprecipitation of Ssop.
Figure 3
Figure 3
Binding of Sec4-N133Ip to Ssop. (A) Binding of overexpressed Sec4 mutant proteins to Ssop. Cells overexpressing wild-type or mutant Sec4 proteins were grown to log phase in YP raffinose media and then shifted to YP galactose media for 6 h before homogenization. An immunoblot from the lysates was probed for Sec4 proteins; anti-Sso immunoprecipitates were probed for coprecipitating Sec4 proteins; and anti-Sncp immunoprecipitates were probed for coprecipitating Ssop. (B) Binding of HA-Sec4-N133Ip to myc-Dss4p, GFP-Sec2p, and Ssop. Cells expressing myc-Dss4p (NY1724) or GFP-Sec2p (NY1723) were mixed with HA-Sec4-N133Ip-expressing cells (NY1710) before homogenization. Myc-Dss4p was immunoprecipitated from the myc-Dss4p + HA-Sec4-N133Ip mixed lysate with anti-myc antibodies. The GFP-Sec2p from the GFP-Sec2p + HA-Sec4-N133Ip mixed lysate was immunoprecipitated with anti-GFP antibodies. For comparison, Ssop was also immunoprecipitated from the GFP-Sec2p + HA-Sec4-N133Ip mixed lysate. Coprecipitating HA-Sec4-N133Ip in the three immunoprecipitates was detected with anti-HA antibodies. HA-Sec4-N133Ip was detectable in the anti-Ssop immunoprecipitate on a longer exposure using the BLAZE detection system. (C) Differential effects in Sec4-N133Ip-overexpressing strains of 2μ DSS4 and SSO plasmids on growth and coprecipitation of Sec4-N133Ip and Sncp with Ssop. Strains were grown for 6 h in YP galactose media before lysis and immunoprecipitation with anti-Ssop antibodies. The immunoprecipitates were probed for coprecipitation of Sec4-N133Ip and Sncp. Suppression of the dominant-negative growth phenotype of Sec4-N133Ip overexpression was measured by observing colony sizes 3 d after streaking on YP galactose plates.
Figure 4
Figure 4
Six nucleotide-free HA-Rab proteins bind to Ssop in lysates. (A) Binding of HA-Rab proteins to Ssop. Strains were grown for 6 h in YP galactose media before homogenization. Anti-Ssop immunoprecipitates and aliquots of the lysates were probed for the HA-Rab proteins. A blot from a second aliquot of the lysate was probed with [α-32P]GTP to detect GTP binding proteins. (B) HA-Sec4-N133Ip binds myc-Ssop in vitro. HA-Sec4-N133Ip and myc-Ssop were either coexpressed in the same cells (NY1719) or expressed in different populations of cells (NY1720 and NY1721) that were mixed before homogenization. An aliquot of the lysates was probed with antibodies against Ssop to detect both myc-Sso2 and native Sso1 and Sso2 proteins and with anti-HA antibodies to detect HA-Sec4-N133Ip. Anti-myc immunoprecipitates were probed to detect coprecipitating HA-Sec4-N133Ip with anti-HA antibodies.
Figure 5
Figure 5
HA-tagged nucleotide-free mutant Rab proteins bind to Pep12p, Sncp, and Ssop but not to SNARE complexes. (A) Nonpreferential binding of nucleotide-free HA-Rab mutant proteins to SNAREs. Expression of the HA-tagged mutant Rab proteins was induced by growth for 6 h in YP galactose media. Lysates were divided into aliquots for immunoprecipitation with antibodies against Pep12p, Sncp, and Ssop. The immunoprecipitates were probed for coprecipitation of the HA-tagged mutant Rab proteins with anti-HA antibody. Similar amounts of untagged Sec4-N133I coimmunoprecipitated with Ssop and Sncp. Thus, the possibility that the lack of specificity we have observed in HA-Rab coprecipitation with SNAREs is an artifact of the N-terminal HA-tag is excluded. (B) HA-Sec4-N133Ip/SNARE binding is insensitive to SNARE complex disassembly. sec18-1 yeast expressing HA-Sec4-N133Ip (NY1718) were either lysed in buffer supplemented with ATP and an ATP-regenerating system or shifted to 37°C for 10 min, collected in ice-cold buffer with NaN3 and NaF, and lysed in buffer containing EDTA. Anti-Snc and anti-Ssop immunoprecipitates were probed with anti HA antibodies for coprecipitating HA-Sec4-N133Ip. Disassembly of the Sncp/Ssop SNARE complex in lysates with ATP was confirmed by probing the anti-Sncp immunoprecipitates with antibodies against Ssop.
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