Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae.
Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
Walworth
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NC
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Brennwald
P
P
Kabcenell
A K
AK
Garrett
M
M
Novick
P
P
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D005656:Fungal Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D005838:Genotype; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D010710:Phosphates; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins