VACUOLAR H-ATPASE OF DICTYOSTELIUM-DISCOIDEUM - A MONOCLONAL-ANTIBODYSTUDY()

A Dictyostelium membrane fraction rich in vacuolar proton pumps, previ ously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 19 91), was used as the immunogen for production of monoclonal antibodies . We obtained antibodies that recognized polypeptides of 100 kDa and 6 8 kDa, corresponding to the two largest subunits of the vacuolar proto n pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of v acuoles and collections of small vesicles typically located just inter nal to the plasma membrane. These vesicles and vacuoles had electron-t ranslucent lumens. No other structures in axenically grown Dictyosteli um cells were labeled to a significant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a monospecific antibody to the B subunit of the chromaffin granule vacuolar ATPase, m arkers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; H euser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two mar kers. Co-localization was seen in both interphase and mitotic cells. T hus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole compl ex in Dictyostelium. In addition, in indirect immunofluorescence exper iments, these monoclonal antibodies provided improved images of the or ganization of the contractile vacuole system.