Endocytosis is an essential process required for key cellular functions such as the uptake of nutrients, recycling of membranes and regulation of signaling pathways. Using a GFP fusion of YP170, a C. elegans yolk protein, we have isolated rme mutants that show defects in receptor-mediated endocytosis. This genetic screen was successful in identifying 11 new genes, including several that encode novel conserved proteins required for endocytic traffic. One such rme mutant,
rme-6 , shows a particularly strong yolk uptake defect in oocytes. In
rme-6 mutants, yolk receptors accumulate to abnormally high levels on the cell surface, suggesting that receptor internalization is blocked.
rme-6 also causes defect in fluid-phase endocytosis in coelomocytes, indicating that
rme-6 is required for at least two classes of endocytosis in at least two cell types. The
rme-6 gene encodes a novel protein that is conserved from worms to humans. RME-6 has a predicted Vps9 domain at its C-terminus and a RasGAP-like domain at its N-terminus. The Vps9 domain is a known motif conserved among guanine nucleotide exchange factors (GEFs) of the small GTPase Rab5. Rab5 is a key regulator of the early endocytic pathway in mammalian cells required for transport from the plasma membrane to the early endosome, and homotypic fusion of early endosomes with one-another.
rme-6 mutants display several phenotypes that specifically indicate low Ce-RAB5 activity, as would be expected if RME-6 functions as a Ce-RAB5 exchange factor. These phenotypes include very small early endosomes and failure to recruit the Rab5 effector EEA1 to early endosomal membranes. Furthermore, double knockdown of
rme-6 and the apparent C. elegans orthologue of the canonical Rab5 exchange factor rabex-5 (Y39A1A.5a) results in complete dispersal of Ce-RAB5 from endosomes to the cytoplasm and loss of embryo viability. A rescuing GFP::
rme-6 transgene is broadly expressed and the GFP::RME-6 fusion protein is enriched in cortical structures of oocytes and coleomocytes, showing significant spatial overlap with the coated-pit protein clathrin and markers of the early endosome. Finally we found that RME-6 interacts physically with the GDP-form of Ce-RAB5 in two-hybrid and co-immunoprecipitation assays. These results demonstrate that RME-6 is a new regulator of Rab5 and indicate that it is a key regulator of endocytosis in metazoans.