Isolation and characterization of effector-loop mutants of CDC42 in yeast.
The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.
Gladfelter
A S
AS
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Moskow
J J
JJ
Zyla
T R
TR
Lew
D J
DJ
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D005800:Genes, Fungal; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C073129:BEM1 protein, S cerevisiae; C095212:CLA4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae