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. In particular, little is known about the proteins required for endocytic recycling, a critical membrane trafficking event in all cells. Functional characterization of a new endocytosis gene,
rme-1 , by C. elegans genetics, suggests that it functions in endocytic recycling.
rme-1 mutants display endocytosis defects in several cell types. These defects include strongly reduced uptake of yolk proteins by oocytes, a receptor-mediated process. In
rme-1 mutants, yolk receptors accumulate in an abnormally expanded cortical endosomal compartment, implicated in recycling.
rme-1 mutant intestinal cells display an abnormal accumulation of basolateral, but not apical, fluid-phase endocytosis markers in large vacuolated structures. Uptake and trafficking of the membrane dye FM 4-64 from the basolateral membranes of the intestine is unaffected by
rme-1 mutations, suggesting that membrane internalization and late endosomal trafficking are independent of
rme-1 . RME-1 is a member of a new class of proteins, evolutionarily conserved among multicellular organisms, bearing an amino-terminal P-loop domain, a central coiled-coil domain, and a carboxy-terminal EH domain. Yeast two-hybrid analysis indicates that the P-loop and coiled-coil regions are required for multimerization. RME-1 is a cytoplasmic protein associated with the periphery of endocytic organelles, consistent with a direct role for RME-1 in endocytic trafficking. We find that Ce-RME-1 and mammalian Rme-1 (mRme-1) expressed in CHO cells localizes primarily to the endocytic-recycling compartment (ERC). In cells expressing dominant negative mRme-1 G429R, a mutation near the EH domain equivalent to a worm dominant negative
rme-1 mutation, we identified a kinetic slowing of transferrin (Tf) recycling with no apparent alteration of the rate constant for Tf internalization. In addition, TGN38 delivery to the TGN from the ERC was slowed in mRme-1 G429R expressing cells. Together, these data suggest that RME-1 functions specifically in the export of molecules from the ERC.