Figure 8.

The cdc37-1 mutation reduces ProA-Mps1p kinase activity. (a) Wild-type (AS231-2d, wt) and mutant (AS231-2b, cdc37-1; Table I) strains carrying the protein A–tagged allele of MPS1 were cultured at 25°C, and some cells were shifted to 38°C for 1 h. Cells were lysed, ProA-Mps1p was immunoprecipitated and assayed for autophosphorylation activity, and kinase activity was normalized for the amount of ProA-Mps1p present in the lysates (Materials and Methods). Autophosphorylation signal is reduced in the cdc37-1 strain even at 25°C (two- to threefold lower activity than wild-type), and drops further after incubation at the nonpermissive temperature (four- to fivefold less than wild-type; 1 h 38°C). Normalized activity values from four independent assays were averaged for each strain and temperature. (b) Cell cycle distribution in each culture was monitored by flow cytometry. The reduction in kinase activity seen after 1 h at 38°C occurs before any G1 bias has appeared in the cdc37-1 culture. The x-axis is the relative DNA content determined by propidium iodide fluorescence, and the y-axis is the relative number of cells (Materials and Methods). Each sample represents 10,000 cells.