Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p.
A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.
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Department of Molecular Biophysics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
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D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D002458:Cell Fractionation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C416529:GYP1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins