NIH3T3 Cells Transfected with the Yeast H+-ATPase Have Altered Rates of Protein Turnover

Abstract

NIH3T3 cells transfected with the yeast plasma membrane H+-ATPase (RN1a line) or transfected with a low-activity mutant H+-ATPase (N-Mut line) were used to examine the relationship between cytosolic pH (pHcyt) and protein turnover. At an extracellular pH (pHex) of 7.15, NIH3T3 and N-Mut cells have a pHcyt of 7-7.1 and a vacuolar pH (pHvac) of 6.3, whereas in RN1a cells both the pHcyt and the pHvac are 0.3 unit more alkaline. Rates of protein synthesis and degradation are optimum at pHex 7.2 and are much more sensitive to pH changes in RN1a cells than in NIH3T3 cells. However, irrespective of pH, rates of protein degradation in RN1a cells are always less than those measured in NIH3T3 cells. Rates of protein synthesis are the same for sparse cultures of RN1a and NIH3T3 cells and show a density-dependent decline in NIH3T3 cells but remain high in RN1a cells even at high cell densities. These data indicate that the elevation of pHcyt caused by transformation with the H+-ATPase has no direct effect on protein synthesis. On the other hand, rates of protein degradation are consistently lower in RN1a cells than in NIH3T3 or N-Mut cells. Basal rates of protein degradation, measured in medium containing 10 mM 3-methyladenine or 10% serum or 1 μM insulin, as well as the autophagic response to serum or insulin withdrawal, are both significantly lower in RN1a cells. These data indicate that transformation with the H+-ATPase has a direct effect on rates of protein degradation, possibly through an elevation of pH. The higher pHvac will directly effect lysosomal protein breakdown and the higher pHcyt may be permissive for maintenance of low basal rates of protein breakdown. Overall, we conclude that transformation with the H+-ATPase provides a permissive environment for high rates of protein synthesis and low rates of protein degradation that result in high rates of growth and the tumor phenotype.