ANTISENSE RNA INHIBITION OF THE PUTATIVE VACUOLAR H-ATPASE PROTEOLIPID OF DICTYOSTELIUM REDUCES INTRACELLULAR CA2+ TRANSPORT AND CELL VIABILITY()

Transport of Ca2+ via a P-type pump into the contractile vacuole of Di ctyostelium discoideum appears to be facilitated by vacuolar proton (V -H+) ATPase activity, To investigate the involvement of the V-Hf-ATPas e in this process using molecular techniques, we cloned a cDNA (vatP) encoding the putative proteolipid subunit of this enzyme. The deduced protein product of this cDNA is composed of 196 amino acids with a cal culated M(r) of 20,148 and the primary structure exhibits high amino a cid sequence identity with V-H+-ATPase proteolipids from other organis ms, vatP is a single-copy gene and it produces one similar to 900 nt t ranscript at relatively constant levels during growth and development. Attempts to disrupt the endogenous gene using vatP cDNA were unsucces sful, But, expression of vatP antisense RNA reduced the levels of vatP message and V-H+-ATPase activity by 50% or more, These antisense stra ins grew and developed slowly, especially under acidic conditions, and the cells seemed to have difficulty forming acidic vesicles, During p rolonged cultivation, all of the antisense strains either reverted to a wild-type phenotype or died, Thus in Dictyostelium, unlike yeast, th e V-H+-ATPase seems to be indispensable for cell viability, When diffe rent antisense strains were analyzed for Ca2+ uptake by the contractil e vacuole, they all accumulated less Ca2+ than control transformants. These results are consistent with earlier pharmacological studies whic h suggested that the V-H+-ATPase functions in intracellular Ca2+ trans port in this organism.