Besides defects in the kinetochore-microtubule attachment reported by Cheng and Chen, cdc48-3 has been shown to be impaired in G1 progression, spindle disassembly at the end of mitosis, transcription factor remodeling, UV-induced turnover of RNAPolII, ERAD, and autophagy. As long as specific targets of Cdc48 at the kinetochore remain unknown, it is therefore almost impossible to differentiate between direct and secondary effects 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 Shp1 leads to the loss of Glc7 accumulation in the nucleus, our microscopy data of strains expressing a fully functional Glc7GFP fusion protein as the sole source of Glc7 indicated only a moderate reduction of nuclear Glc7 in shp1. These data are supported by a normal co-immunoprecipitation of Glc7 with its nuclear targeting subunit Sds22 in shp1, and they are in agreement with data from biochemical fractionation experiments. There are two potential explanations for the discrepancy of our data with those by Cheng and Chen. First, we found that the nuclear localization of Glc7GFP in shp1 is reduced in the presence of additional, untagged Glc7 for unknown reasons. Cheng and Chen used a strain expressing GFPGlc7 in addition to endogenous Glc7, raising the possibility that these conditions prevented a nuclear localization of the tagged Glc7 variant. Second, Cheng and Chen performed microscopy 12 hours after promoter shut-off under conditions of ongoing cell death, whereas our analysis was performed with logarithmically growing shp1 cells. Altogether, considering the available experimental evidence, a gross reduction of nuclear Glc7 levels in shp1 null mutants appears unlikely. In line with this conclusion, cytoplasmic Glc7 functions in glycogen metabolism and in the Vid pathway are affected in shp1 mutants as well, also arguing against impaired nuclear localization of Glc7 as the critical defect in shp1. Besides the genetic interactions between glc7 and shp1 mutants, the present study showed for the first time that Shp1 and Glc7 also interact physically. We currently do not know if this interaction is direct or indirect, for instance bridged by regulatory subunits of Glc7. While Shp1 lacks a classical RVxF motif, which mediates the binding of many PP1 regulatory subunits, a number of Glc7 subunits interact through other motifs. Alternatively, Cdc48Shp1 could interact with ubiquitylated Glc7 or an ubiquitylated Glc7 interactor. Consistent with this possibility, we found that Glc7 is ubiquitylated in vivo, in agreement with proteomics studies.

www.bioactivescreeninglibrary.com has actually always been our primary interest at http://www.mapkangiogenesis.com/index.php/2019/02/22/measured-glucose-consumption-lactate-generation-bcap-37-cells-incubated-medium/: find out more concerning exactly how all of it began on our internet site.