Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis.
The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.
Cell and Molecular Biology Program Research Program in Structural Biology and Biophysics, Institute of Biotechnology, FI-0001 University of Helsinki, Finland.
Marion
M
Weber-Boyvat
Nina
N
Aro
Konstantin G
KG
Chernov
Tuula
T
Nyman
Jussi
J
Jäntti
D000595:Amino Acid Sequence; D005089:Exocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D050600:SNARE Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; C106953:MSO1 protein, S cerevisiae; D008565:Membrane Proteins; D051938:Munc18 Proteins; C497931:SEC1 protein, S cerevisiae; C064456:SEC2 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins