Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p).
Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.
Jaquenoud
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M
Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD, Switzerland.
Peter
M
M
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D002453:Cell Cycle; D002850:Chromatography, Gel; D017124:Conserved Sequence; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D005799:Genes, Dominant; D008840:Microfilament Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C107727:BUD6 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; C109253:GIC2 protein, S cerevisiae; D008840:Microfilament Proteins; C065889:SPA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; D053778:Thiazolidines; D020764:cdc42 GTP-Binding Protein; C037067:latrunculin A