A RAB4-LIKE GTPASE IN DICTYOSTELIUM-DISCOIDEUM COLOCALIZES WITH V-H-ATPASES IN RETICULAR MEMBRANES OF THE CONTRACTILE VACUOLE COMPLEX AND IN LYSOSOMES()

In the course of screening a cDNA library for ras-related Dictyosteliu m discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small M(r) GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot a nalysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expresse d at relatively constant levels during growth and differentiation. Aff inity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polyp eptide on western blots of cell extracts. Density gradient fractionati on revealed that the RabD antigen co-distributed primarily with buoyan t membranes rich in vacuolar proton pumps (V-H+-ATPases) and, to a les ser extent, with lysosomes. This result was confirmed by examining cel l lines expressing an epitope-tagged version of RabD. Magnetically pur ified early endocytic vesicles and post-lysosomal vacuoles reacted mor e weakly with anti-RabD antibodies than did lysosomes. Other organelle s were negative for RabD. Double-label indirect immunofluorescence mic roscopy revealed that RabD and the 100 kDa V-H+-ATPase subunit colocal ized in a fine reticular network throughout the cytoplasm. This networ k was reminiscent of spongiomes, the tubular elements of the contracti le vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H+-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally as sociated with the spongiomes of contractile vacuole complex.