NIH3T3 CELLS TRANSFECTED WITH THE YEAST H-ATPASE HAVE ALTERED RATES OF PROTEIN-TURNOVER()

NIH3T3 cells transfected with the yeast plasma membrane H+-ATPase (RN1 a line) or transfected with a low-activity mutant H+-ATPase (N-Mut lin e) were used to examine the relationship between cytosolic pH (pH(cyt) ) and protein turnover. At an extracellular pH (pH(cyt)) of 7.15, NIH3 T3 and N-Mut cells have a pH(cyt) of 7-7.1 and a vacuolar pH (pH(vac)) Of 6.3, whereas in RN1a cells both the pH(cyt) and the pH(vac) are 0. 3 unit more alkaline. Rates of protein synthesis and degradation are o ptimum at pH(ex) 7.2 and are much more sensitive to pH changes in RN1a cells than in NIH3T3 cells. However, irrespective of pH, rates of pro tein degradation in RN1a cells are always less than those measured in NIH3T3 cells. Rates of protein synthesis are the same for sparse cultu res of RN1a and NIH3T3 cells and show a density-dependent decline in N IH3T3 cells but remain high in RN1a cells even at high cell densities. These data indicate that the elevation of pH(cyt) caused by transform ation with the H+-ATPase has no direct effect on protein synthesis. On the other hand, rates of protein degradation are consistently lower i n RN1a cells than in NIH3T3 or N-Mut cells. Basal rates of protein deg radation, measured in medium containing 10 mM 3-methyladenine or 10% s erum or 1 mu M 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 eff ect on rates of protein degradation, possibly through an elevation of pH. The higher pH(vac) will directly effect lysosomal protein breakdow n and the higher pH(cyt) may be permissive for maintenance of low basa l rates of protein breakdown. Overall, we conclude that transformation with the H+-ATPase provides a permissive environment for high rates o f protein synthesis and low rates of protein degradation that result i n high rates of growth and the tumor phenotype. (C) 1994 Academic Pres s, Inc.