Figure 5.

Mislocalization of Sky1p to the nucleus inhibits growth. (A) Domain arrangement of wild-type SKY1, mutant sky1 with the spacer domain precisely deleted [sky1(Δspacer)], and sky1(Δspacer) that also carries a K-to-M point mutation (kinase-inactivating) at the ATP binding site. (B) Immunofluorescent localization of the indicated kinases, expressed as N-terminal fusion proteins with the FLAG epitope, using a monoclonal anti-FLAG antibody (b, d, and f). DNA was stained with 4′,6-diamidino-2-phenylindole to reveal the positions of the nuclei (a, c, and e). (C) Growth phenotypes associated with Sky1p overexpression. Overexpression from the GAL1 promoter of the indicated wild-type or mutant forms of Sky1p was induced in wild-type cells streaked onto plates containing galactose. Cells containing the plasmid vector alone (pYES) served as a control. (D) The indicated forms of Sky1p were immunoprecipitated from yeast extracts before (lanes 2 and 3) or after (lanes 5 and 6) induction of their expression by galactose and then were tested for kinase activity by using ASF/SF2 as the substrate. Cells containing the plasmid vector alone (pYES) served as a control.