Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.
Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.
Poon
P P
PP
Department of Microbiology and Immunology, Dalhousie University, Halifax, NS Canada.
Wang
X
X
Rotman
M
M
Huber
I
I
Cukierman
E
E
Cassel
D
D
Singer
R A
RA
Johnston
G C
GC
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006026:Glycoside Hydrolases / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012969:Sodium Fluoride / Q000494:pharmacology; D016335:Zinc Fingers; D043324:beta-Fructofuranosidase
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C089147:GCS1 protein, S cerevisiae; D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012969:Sodium Fluoride; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors