For the expression of the indicated wild-type and mutant SHP1 and

For the expression of the indicated wild-type and mutant SHP1 and GLC8 alleles were analyzed as described in the legend to Fig. 4a. (c, d, e) Reduced physical interaction between Glc7 and Glc8 in shp1-7. Lysates of asynchronous (c) or a-factor-arrested (d) wild-type and shp17 cells expressing Glc83HA were subjected to immunoprecipitation with anti-HA antibody and analyzed for co-precipitation of endogenous, untagged Glc7. The asterisks mark a cross-reactive band of the Glc7 antibody 1655472 (Glc7 blots) and the immunoglobulin light chain of the HA antibody (Glc8 blots), respectively. In panel (c), irrelevant lanes were removed from the figure. However, all lanes shown were on the same Western blot and exposed and PS 1145 biological activity processed identically. (e) Quantification of three independent experiments as in panel (c), showing the ratio of the Glc7 and Glc8 signal intensities. (f) Overexpression of GLC8 partially suppresses the temperature sensitivity of shp1-7. Wild-type (WT) and shp1-7 cells expressing the indicated GLC83HA alleles from an integrative plasmid under the control of the inducible PMET25 promoter were analyzed for growth at the indicated temperatures in the presence (+Met (off)) and absence (2Met (on)) of methionine in the growth medium. doi:10.1371/journal.pone.0056486.gdiffers in certain central aspects. We were able to demonstrate strong positive and negative genetic interactions, respectively, of shp1 null and Cdc48 binding-deficient alleles with ipl1-321 and glc7-129. Importantly, we proved that the cell cycle and chromosome segregation defects of shp1 null and Cdc48 bindingdeficient mutants are efficiently suppressed by increased Glc7 levels. Finally, we established an increased Dam1 phosphorylation in shp1 mutants, which can be suppressed by a reduction of Ipl1 activity. One likely explanation for the differences between the two PD1-PDL1 inhibitor 1 site studies relates to the strains used by Cheng and Chen. In particular, the use of the cdc48-3 strain poses problems due to its pleiotropic phenotypes. Besides defects in the kinetochore-microtubule attachment reported by Cheng and Chen, cdc48-3 has been shown to be impaired in G1 progression [64,66], spindle disassembly at the end of mitosis [65], transcription factor remodeling [102], UV-induced turnover of RNAPolII [24], ERAD [103,104], and autophagy [30]. As long as specific targets of Cdc48 at the kinetochore remain unknown, it is therefore almost impossible to differentiate between direct and secondaryeffects of the cdc48-3 allele on cell cycle progression. Furthermore, Cheng and Chen state that the observed mitotic phenotypes of cdc48-3 were generally more severe than those of Shp1-depleted cells. This finding is likely to reflect the involvement of alternative Cdc48 cofactors, in particular Ufd1-Npl4, in Shp1-independent functions of Cdc48 during the cell cycle. Taken together, the uncertainties in the interpretation of cdc48-3 phenotypes underscore the importance of designing specific Cdc48 binding-deficient shp1 alleles. The shp1 alleles presented in this study enabled us to study genetic interactions and the effect of GLC7 over-expression in the absence of unrelated pleiotropic defects and thus allowed us to formally conclude for the first time that the regulation of Glc7 activity indeed requires the Cdc48Shp1 complex. The major discrepancy between this study and the study by Cheng and Chen relates to the cellular localization of Glc7 in the absence of Shp1. While these authors found that depletion of Sh.For the expression of the indicated wild-type and mutant SHP1 and GLC8 alleles were analyzed as described in the legend to Fig. 4a. (c, d, e) Reduced physical interaction between Glc7 and Glc8 in shp1-7. Lysates of asynchronous (c) or a-factor-arrested (d) wild-type and shp17 cells expressing Glc83HA were subjected to immunoprecipitation with anti-HA antibody and analyzed for co-precipitation of endogenous, untagged Glc7. The asterisks mark a cross-reactive band of the Glc7 antibody 1655472 (Glc7 blots) and the immunoglobulin light chain of the HA antibody (Glc8 blots), respectively. In panel (c), irrelevant lanes were removed from the figure. However, all lanes shown were on the same Western blot and exposed and processed identically. (e) Quantification of three independent experiments as in panel (c), showing the ratio of the Glc7 and Glc8 signal intensities. (f) Overexpression of GLC8 partially suppresses the temperature sensitivity of shp1-7. Wild-type (WT) and shp1-7 cells expressing the indicated GLC83HA alleles from an integrative plasmid under the control of the inducible PMET25 promoter were analyzed for growth at the indicated temperatures in the presence (+Met (off)) and absence (2Met (on)) of methionine in the growth medium. doi:10.1371/journal.pone.0056486.gdiffers in certain central aspects. We were able to demonstrate strong positive and negative genetic interactions, respectively, of shp1 null and Cdc48 binding-deficient alleles with ipl1-321 and glc7-129. Importantly, we proved that the cell cycle and chromosome segregation defects of shp1 null and Cdc48 bindingdeficient mutants are efficiently suppressed by increased Glc7 levels. Finally, we established an increased Dam1 phosphorylation in shp1 mutants, which can be suppressed by a reduction of Ipl1 activity. One likely explanation for the differences between the two studies relates to the strains used by Cheng and Chen. In particular, the use of the cdc48-3 strain poses problems due to its pleiotropic phenotypes. Besides defects in the kinetochore-microtubule attachment reported by Cheng and Chen, cdc48-3 has been shown to be impaired in G1 progression [64,66], spindle disassembly at the end of mitosis [65], transcription factor remodeling [102], UV-induced turnover of RNAPolII [24], ERAD [103,104], and autophagy [30]. As long as specific targets of Cdc48 at the kinetochore remain unknown, it is therefore almost impossible to differentiate between direct and secondaryeffects of the cdc48-3 allele on cell cycle progression. Furthermore, Cheng and Chen state that the observed mitotic phenotypes of cdc48-3 were generally more severe than those of Shp1-depleted cells. This finding is likely to reflect the involvement of alternative Cdc48 cofactors, in particular Ufd1-Npl4, in Shp1-independent functions of Cdc48 during the cell cycle. Taken together, the uncertainties in the interpretation of cdc48-3 phenotypes underscore the importance of designing specific Cdc48 binding-deficient shp1 alleles. The shp1 alleles presented in this study enabled us to study genetic interactions and the effect of GLC7 over-expression in the absence of unrelated pleiotropic defects and thus allowed us to formally conclude for the first time that the regulation of Glc7 activity indeed requires the Cdc48Shp1 complex. The major discrepancy between this study and the study by Cheng and Chen relates to the cellular localization of Glc7 in the absence of Shp1. While these authors found that depletion of Sh.