Identification of novel mutations in ACT1 and SLA2 that suppress the actin-cable-overproducing phenotype caused by overexpression of a dominant active form of Bni1p in Saccharomyces cerevisiae.
A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1DeltaN). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1Delta. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PFY1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82 were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1Delta sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1DeltaN phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.
Yoshiuchi
Shiro
S
Division of Molecular Interaction, Institute of Genetic Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-0815, Japan.
Yamamoto
Takaharu
T
Sakane
Hiroshi
H
Kadota
Jun
J
Mochida
Junko
J
Asaka
Masahiro
M
Tanaka
Kazuma
K
D000199:Actins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D003598:Cytoskeletal Proteins; D015870:Gene Expression; D005799:Genes, Dominant; D005800:Genes, Fungal; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D051304:Profilins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
C465210:Act1 protein, S cerevisiae; D000199:Actins; C107727:BUD6 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; C086662:CDC3 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D051304:Profilins; C082266:SLA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins