J. Bush et al., A ROLE FOR A RAB4-LIKE GTPASE IN ENDOCYTOSIS AND IN REGULATION OF CONTRACTILE VACUOLE STRUCTURE AND FUNCTION IN DICTYOSTELIUM-DISCOIDEUM, Molecular biology of the cell, 7(10), 1996, pp. 1623-1638
The small M(r) Rab4-like GTPase, RabD, localizes to the endosomal path
way and the contractile vacuole membrane system in Dictyostelium disco
ideum. Stably transformed cell lines overexpressing a dominant negativ
e functioning RabD internalized fluid phase marker at 50% of the rate
of wild-type cells. Mutant cells were also slower at recycling interna
lized fluid. Microscopic and biochemical approaches indicated that the
transport of fluid to large postlysosome vacuoles was delayed in muta
nt cells, resulting in an accumulation in acidic smaller vesicles, pro
bably lysosomes. Also, RabD N121I-expressing cell lines missorted a sm
all but significant percentage of newly synthesized lysosomal alpha-ma
nnosidase precursor polypeptides. However, the majority of the newly s
ynthesized alpha-mannosidase was transported with normal kinetics and
correctly delivered to lysosomes. Subcellular fractionation and immuno
fluorescent microscopy indicated that in mutant cells contractile vacu
ole membrane proteins were associated with compartments morphologicall
y distinct from the normal reticular network. Osmotic tests revealed t
hat the contractile vacuole functioned inefficiently in mutant cells.
Our results suggest that RabD regulates membrane traffic along the end
osomal pathway, and that this GTPase may play a role in regulating the
structure and function of the contractile vacuole system by facilitat
ing communication with the endosomal pathway.