| Abstract: | The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins. |
