Ted in Cdc48 bindingdeficient shp1 alleles are indicated in bold in

Ted in Cdc48 bindingdeficient shp1 alleles are indicated in bold in the sequence and by asterisks in the outlines of the Shp1 variant proteins shown below. (b) Simultaneous mutation of the R-FPR motif in the UBX domain and of binding site(s) 1 abolishes Cdc48 binding in vivo. Lysates of cells expressing the indicated shp1 alleles were subjected to immunoprecipitation with a Shp1 antibody and analyzed for Cdc48 co-immunoprecipitation by Western blot. (c) shp1 mutants defective in Cdc48 binding are temperature sensitive. Wild-type (WT) and shp1-7 mutant cells carrying the indicated centromeric plasmids were analyzed for growth at the indicated temperatures as described for Fig. 1a. (d, e) shp1 mutants defective in Cdc48 binding are delayed in mitotic progression. Asynchronous WT and shp1-a1 cultures were analyzed by FACS (d) as described in the legend to Fig. 1b, and WTRegulation 23727046 of Glc7 by Cdc48Shpand shp1-a1 cultures synchronized by a-factor arrest/release were analyzed by Western blot against the mitotic cyclin Clb2 (e) as described in the legend to Fig. 1d. doi:10.1371/CASIN site journal.pone.0056486.ganalyzed genetic interactions between shp1 and the conditional glc7 allele, glc7-129, which at the non-permissive temperature confers a cell cycle arrest at the metaphase-anaphase transition [76]. Furthermore, the mitotic arrest of glc7-129 was reported toFigure 3. The cell cycle delay of shp1 mutants is caused by SAC activation. (a) Delayed degradation of Pds1 (securin). Wild-type (WT) and shp1-7 null mutant cultures were synchronized by a-factor arrest/ release and analyzed by Western blot against Pds118myc as described in the legend to Fig. 1d. (b) shp1-7 is hypersensitive towards the spindle poison benomyl. Growth of WT, shp1-7 and Dmad2 cells at 25uC in the absence (DMSO) and presence of benomyl was analyzed as described for Fig. 1a. (c) Synthetic growth defect of shp1-7 Dmad2. Haploid progeny of one tetrad from the cross of shp1-7 with Dmad2 was analyzed for growth at 25uC as described for Fig. 1a. (d) The mitotic delay of shp1-7 is alleviated by checkpoint inactivation. The cell cycle distribution of the indicated strains at 25uC was analyzed by FACS as described in the legend to Fig. 1b. doi:10.1371/journal.pone.0056486.gdepend on the SAC [49]. Intriguingly, the shp1-7 glc7-129 double mutant was inviable at all temperatures tested (Fig. S1a and data not shown), indicating overlapping cellular functions of Shp1 and Glc7. As expected, the synthetic lethality of shp1-7 glc7-129 could be suppressed by a centromeric plasmid encoding wild-type SHP1 (Fig. 4a). Importantly, when we tested the Cdc48 binding-deficient alleles shp1DUBX, shp1-a1, and shp1-b1, their Mirin site ability to suppress the lethality of shp1-7 glc7-129 correlated with the ability of the respective gene products to bind Cdc48, demonstrating that an intact Cdc48Shp1 complex is required for the viability of glc7-129. To confirm that Shp1 is involved in mitotic functions of Glc7, we next tested genetic interactions between SHP1 and the major nuclear Glc7 regulatory subunit, SDS22 (Fig. 4b). Indeed, we observed synthetic lethality of the shp1-7 sds22-6 double mutant as well, strongly suggesting that Shp1 is critical for a mitotic function(s) of Glc7. Finally, we analyzed genetic interactions between SHP1 and IPL1, the gene encoding the yeast Aurora B kinase homologue. Ipl1 has been described to antagonize mitotic functions of Glc7 at the kinetochore, and the correct balance of Ipl1 kinase and.Ted in Cdc48 bindingdeficient shp1 alleles are indicated in bold in the sequence and by asterisks in the outlines of the Shp1 variant proteins shown below. (b) Simultaneous mutation of the R-FPR motif in the UBX domain and of binding site(s) 1 abolishes Cdc48 binding in vivo. Lysates of cells expressing the indicated shp1 alleles were subjected to immunoprecipitation with a Shp1 antibody and analyzed for Cdc48 co-immunoprecipitation by Western blot. (c) shp1 mutants defective in Cdc48 binding are temperature sensitive. Wild-type (WT) and shp1-7 mutant cells carrying the indicated centromeric plasmids were analyzed for growth at the indicated temperatures as described for Fig. 1a. (d, e) shp1 mutants defective in Cdc48 binding are delayed in mitotic progression. Asynchronous WT and shp1-a1 cultures were analyzed by FACS (d) as described in the legend to Fig. 1b, and WTRegulation 23727046 of Glc7 by Cdc48Shpand shp1-a1 cultures synchronized by a-factor arrest/release were analyzed by Western blot against the mitotic cyclin Clb2 (e) as described in the legend to Fig. 1d. doi:10.1371/journal.pone.0056486.ganalyzed genetic interactions between shp1 and the conditional glc7 allele, glc7-129, which at the non-permissive temperature confers a cell cycle arrest at the metaphase-anaphase transition [76]. Furthermore, the mitotic arrest of glc7-129 was reported toFigure 3. The cell cycle delay of shp1 mutants is caused by SAC activation. (a) Delayed degradation of Pds1 (securin). Wild-type (WT) and shp1-7 null mutant cultures were synchronized by a-factor arrest/ release and analyzed by Western blot against Pds118myc as described in the legend to Fig. 1d. (b) shp1-7 is hypersensitive towards the spindle poison benomyl. Growth of WT, shp1-7 and Dmad2 cells at 25uC in the absence (DMSO) and presence of benomyl was analyzed as described for Fig. 1a. (c) Synthetic growth defect of shp1-7 Dmad2. Haploid progeny of one tetrad from the cross of shp1-7 with Dmad2 was analyzed for growth at 25uC as described for Fig. 1a. (d) The mitotic delay of shp1-7 is alleviated by checkpoint inactivation. The cell cycle distribution of the indicated strains at 25uC was analyzed by FACS as described in the legend to Fig. 1b. doi:10.1371/journal.pone.0056486.gdepend on the SAC [49]. Intriguingly, the shp1-7 glc7-129 double mutant was inviable at all temperatures tested (Fig. S1a and data not shown), indicating overlapping cellular functions of Shp1 and Glc7. As expected, the synthetic lethality of shp1-7 glc7-129 could be suppressed by a centromeric plasmid encoding wild-type SHP1 (Fig. 4a). Importantly, when we tested the Cdc48 binding-deficient alleles shp1DUBX, shp1-a1, and shp1-b1, their ability to suppress the lethality of shp1-7 glc7-129 correlated with the ability of the respective gene products to bind Cdc48, demonstrating that an intact Cdc48Shp1 complex is required for the viability of glc7-129. To confirm that Shp1 is involved in mitotic functions of Glc7, we next tested genetic interactions between SHP1 and the major nuclear Glc7 regulatory subunit, SDS22 (Fig. 4b). Indeed, we observed synthetic lethality of the shp1-7 sds22-6 double mutant as well, strongly suggesting that Shp1 is critical for a mitotic function(s) of Glc7. Finally, we analyzed genetic interactions between SHP1 and IPL1, the gene encoding the yeast Aurora B kinase homologue. Ipl1 has been described to antagonize mitotic functions of Glc7 at the kinetochore, and the correct balance of Ipl1 kinase and.