The endocytic pathway is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. The steps involved in uptake and endocytic trafficking within the endosomal system have been described, but many of the components mediating these steps at the molecular level remain to be identified. We have identified a group of genes required for endocytosis by developing oocytes, the rme genes. Many of these genes are required for endocytosis in multiple cell-types and have close relatives in humans that were not previously implicated in endocytosis. Recently we have focused on
rme-6, an endocytosis gene represented by eleven viable alleles.
rme-6 mutants accumulate yolk proteins in the pseudocoelom and have greatly reduced yolk accumulation by oocytes, indicating a defect in receptor-mediated endocytosis.
rme-6 mutants appear to have elevated levels of cell-surface yolk receptors, consistent with a defect in yolk receptor internalization. We also found that
rme-6 mutants display severe defects in the uptake of pseudocoelomic fluid-phase markers by coelomocytes, scavenger cells of the body cavity. We were able to visualize pre-lysosomal endosomes of the coelomocyte using an RME-8::GFP marker. Microinjection of Texas-Red BSA (TR-BSA) into the pseudocoelom allowed us to follow endocytosis and intracellular transport of TR-BSA within the coelomocytes over time.
rme-6 mutants displayed unusually small RME-8::GFP labeled endosomes. We also noted that accumulation of TR-BSA in these endosomes was delayed in
rme-6 mutants, but that TR-BSA accumulation in labeled endosomes did occur in
rme-6 mutants, and export of TR-BSA from the endosomes to presumed lysosomes also occurred. These results suggest a role for RME-6 early in endocytosis, either in internalization and/or endosome fusion events. Molecular analysis of RME-6 indicates that it contains an N-terminal RAS-GAP-like domain and a C-terminal Rabex-5-like domain. Both of these motifs suggest that RME-6 exerts its influence on endocytosis by regulating the activity of small GTPases, perhaps RAS and/or RAB-5. This combination of domains also suggests the intriguing possibility that RME-6 links signal transduction processes with endocytic trafficking. In the coelomocyte an GFP:: RME-6 fusion is cytoplasmic, and accumulates in zones near the plasma membrane, consistent with a direct role in early endocytic events.