Noprecipitation with Glc8 in asynchronous cultures of wild-type and shp1-

Noprecipitation with Glc8 in asynchronous cultures of wild-type and shp1-7 revealed that the Glc7-Glc8 interaction in shp1-7 was reduced by approximately 50 (Fig. 8e). These data show that Shp1 is Verubecestat required for a normal physical interaction between Glc7 and its activator Glc8. We next tested if the reduced interaction between Glc7 and Glc8 in shp1-7 is responsible for shp1 phenotypes. To this end, we over-expressed GLC83HA under the control of the MET25 promoter (Fig. 8f). Importantly, the over-expression of 25033180 GLC83HA partially TA02 web suppressed the growth defects of shp1-7, as improved growth at 35uC and weak, but detectable growth at the nonpermissive temperature of 37uC was observed (Fig. 8f). In contrast, over-expression of glc8-T118A3HA was toxic in shp1-7 cells (Fig. 8f), presumably because excess non-activating Glc8-T118 protein competed with endogenous Glc8 for Glc7 binding. In summary, our data show that the Cdc48Shp1 complex is important for the activation of Glc7 by Glc8, and that lack of Glc8-mediated activation contributes critically to the phenotype of shp1 mutants.DiscussionThis study addresses the relationship of Shp1, a major Cdc48 cofactor, and Glc7, the catalytic subunit of budding yeast PP1. We found that shp1 mutants exhibit a variety of severe phenotypes, including a significant mitotic delay during progression from metaphase to anaphase. We were able to show that the mitotic phenotype of shp1 mutants is caused by limiting nuclear Glc7 activity towards mitotic substrates, resulting in their hyperphosphorylation due to unbalanced Ipl1 kinase activity. By engineering shp1 alleles specifically defective in Cdc48 binding, we established that Shp1 regulates Glc7 in its capacity as a Cdc48 cofactor. Importantly, we could demonstrate that Shp1 and Glc7 interact physically, and that the Cdc48Shp1 complex is required for normal interaction of Glc7 with Glc8. shp1 mutants were originally found to exhibit reduced Glc7 activity towards glycogen phosphorylase, decreased glycogen accumulation, and defective sporulation [32]. Other shp1 phenotypes attributed to reduced Glc7 activity include defective vacuolar degradation of fructose-1,6-bisphosphatase through the vacuole import and degradation (Vid) pathway [60], impaired V-ATPase activity [61], and impaired glucose repression [62]. Here, we provide several lines of evidence that shp1 mutants also possesses a significant defect in mitotic Glc7 activity. First, the genetic interactions between shp1 and glc7, sds22, mad2, and ipl1 all point towards impaired nuclear 1317923 function(s) of Glc7 in shp1. Second, overexpression of GLC7 in shp1 restored a normal cell cycle distribution and suppressed chromosome segregation defects. Third, the nuclear Glc7 substrates histone H3 and Dam1 are hyperphosphorylated in shp1 in an Ipl1-dependent manner. Together with the previously described cytosolic and vacuolar processes, the elucidation of its involvement in mitotic Glc7 functions underFigure 6. Dam1 hyper-phosphorylation is critical for the impaired growth of shp1. (a) Hyper-phosphorylation of Dam1 in shp1-7. The phosphorylation state of Dam19myc in the indicated wild-type (WT) and mutant strains at 35uC was analyzed by Western blot against the myc epitope tag. The position of Dam1 and phosphorylated Dam1 (Dam1-P) is indicated. (b) Phosphorylation-deficient and phosphorylation-mimicking dam1 mutants suppress and exacerbate, respectively, the growth phenotype of shp1-7. Growth of WT and shp1-7 cells carryin.Noprecipitation with Glc8 in asynchronous cultures of wild-type and shp1-7 revealed that the Glc7-Glc8 interaction in shp1-7 was reduced by approximately 50 (Fig. 8e). These data show that Shp1 is required for a normal physical interaction between Glc7 and its activator Glc8. We next tested if the reduced interaction between Glc7 and Glc8 in shp1-7 is responsible for shp1 phenotypes. To this end, we over-expressed GLC83HA under the control of the MET25 promoter (Fig. 8f). Importantly, the over-expression of 25033180 GLC83HA partially suppressed the growth defects of shp1-7, as improved growth at 35uC and weak, but detectable growth at the nonpermissive temperature of 37uC was observed (Fig. 8f). In contrast, over-expression of glc8-T118A3HA was toxic in shp1-7 cells (Fig. 8f), presumably because excess non-activating Glc8-T118 protein competed with endogenous Glc8 for Glc7 binding. In summary, our data show that the Cdc48Shp1 complex is important for the activation of Glc7 by Glc8, and that lack of Glc8-mediated activation contributes critically to the phenotype of shp1 mutants.DiscussionThis study addresses the relationship of Shp1, a major Cdc48 cofactor, and Glc7, the catalytic subunit of budding yeast PP1. We found that shp1 mutants exhibit a variety of severe phenotypes, including a significant mitotic delay during progression from metaphase to anaphase. We were able to show that the mitotic phenotype of shp1 mutants is caused by limiting nuclear Glc7 activity towards mitotic substrates, resulting in their hyperphosphorylation due to unbalanced Ipl1 kinase activity. By engineering shp1 alleles specifically defective in Cdc48 binding, we established that Shp1 regulates Glc7 in its capacity as a Cdc48 cofactor. Importantly, we could demonstrate that Shp1 and Glc7 interact physically, and that the Cdc48Shp1 complex is required for normal interaction of Glc7 with Glc8. shp1 mutants were originally found to exhibit reduced Glc7 activity towards glycogen phosphorylase, decreased glycogen accumulation, and defective sporulation [32]. Other shp1 phenotypes attributed to reduced Glc7 activity include defective vacuolar degradation of fructose-1,6-bisphosphatase through the vacuole import and degradation (Vid) pathway [60], impaired V-ATPase activity [61], and impaired glucose repression [62]. Here, we provide several lines of evidence that shp1 mutants also possesses a significant defect in mitotic Glc7 activity. First, the genetic interactions between shp1 and glc7, sds22, mad2, and ipl1 all point towards impaired nuclear 1317923 function(s) of Glc7 in shp1. Second, overexpression of GLC7 in shp1 restored a normal cell cycle distribution and suppressed chromosome segregation defects. Third, the nuclear Glc7 substrates histone H3 and Dam1 are hyperphosphorylated in shp1 in an Ipl1-dependent manner. Together with the previously described cytosolic and vacuolar processes, the elucidation of its involvement in mitotic Glc7 functions underFigure 6. Dam1 hyper-phosphorylation is critical for the impaired growth of shp1. (a) Hyper-phosphorylation of Dam1 in shp1-7. The phosphorylation state of Dam19myc in the indicated wild-type (WT) and mutant strains at 35uC was analyzed by Western blot against the myc epitope tag. The position of Dam1 and phosphorylated Dam1 (Dam1-P) is indicated. (b) Phosphorylation-deficient and phosphorylation-mimicking dam1 mutants suppress and exacerbate, respectively, the growth phenotype of shp1-7. Growth of WT and shp1-7 cells carryin.