The classical nuclear localization signal receptor, importin-alpha, is required for efficient transition through the G1/S stage of the cell cycle in Saccharomyces cerevisiae.
There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-alpha/Srp1, to the G(2)/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-alpha/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G(1) to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G(1)/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G(2)/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-alpha mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G(1)/S cell cycle transition.
Pulliam
Kanika F
KF
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Fasken
Milo B
MB
McLane
Laura M
LM
Pulliam
John V
JV
Corbett
Anita H
AH
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D016193:G1 Phase; D016153:Genes, Suppressor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D028884:Karyopherins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism*
D028884:Karyopherins; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D029701:Saccharomyces cerevisiae Proteins; C538902:Srp1 protein, S cerevisiae; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins