Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast.
SEC4 is an essential gene encoding a small GTPase that is involved in Golgi to cell surface transport in Saccharomyces cerevisiae and is a paradigm for studies on the mode of action of Rab proteins. We describe here the features of interaction of Sec4p with the accessory protein Dss4p. Dss4p is found both on membranes and in the cytosol; however, it is the membrane fraction that is complexed to Sec4p. Dss4p, like its mammalian counterpart, Mss4, binds zinc, and disruption of the zinc-binding site disrupts the ability of the protein to interact with Sec4p. DSS4 overexpression can rescue the lethal phenotype of two alleles of SEC4, corresponding to dominant mutations of Ras. We demonstrate that this suppression is due to the ability of Dss4p to form a tight complex with the mutant forms of Sec4p and hence sequester the mutant protein from its inhibitory effect. These results imply an in vivo role for Dss4p as a guanine nucleotide dissociation stimulator. In vitro the protein has the ability to stimulate the dissociation rate of both GDP and GTP from Sec4p. We examined the relationship of GDI1 and DSS4 with SEC4 both genetically and biochemically. These results exclude a role for DSS4 in the recruitment of Sec4p/GDI onto membranes.
Collins
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RN
Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA.
Brennwald
P
P
Garrett
M
M
Lauring
A
A
Novick
P
P
D000483:Alleles; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005838:Genotype; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins
D017931:DNA Primers; C079548:DSS4 protein, S cerevisiae; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins