A RAB4-LIKE GTPASE IN DICTYOSTELIUM-DISCOIDEUM COLOCALIZES WITH V-H-ATPASES IN RETICULAR MEMBRANES OF THE CONTRACTILE VACUOLE COMPLEX AND IN LYSOSOMES()
J. Bush et al., A RAB4-LIKE GTPASE IN DICTYOSTELIUM-DISCOIDEUM COLOCALIZES WITH V-H-ATPASES IN RETICULAR MEMBRANES OF THE CONTRACTILE VACUOLE COMPLEX AND IN LYSOSOMES(), Journal of Cell Science, 107, 1994, pp. 2801-2812
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.