Endocytosis is essential to many aspects of metazoan life, including nutrient uptake, synaptic vesicle recycling, and growth factor receptor down-regulation. Much remains to be learned about this important cellular pathway. We have isolated a collection of endocytosis defective mutants, the rme genes. Recently, we focused on the analysis of
rme-1, a gene that may regulate endocytic recycling.
rme-1 mutants are defective in endocytosis in multiple cell types including oocytes and coelomocytes.
rme-1 mutants also develop large vacuoles in the intestine and hypodermis. The intestinal vacuoles accumulate endocytosis markers, but only from their basolateral (pseudocoelomic) surfaces. Specifically, we found that fluorescent dextran microinjected into the pseudocoelom rapidly accumulates within
rme-1 mutant vacuoles. Another endocytosis marker microinjected into the pseudocoelom, the membrane dye FM4-64, is excluded from
rme-1 mutant vacuoles and instead accumulates in autofluorescent gut granules (lysosomes). Because fluid-phase markers like dextran can enter the recycling pathway, and FM4-64 does not,
rme-1 mutant vacuoles may represent aberrant recycling endosomes. We found that
rme-1 encodes a member of a new family of proteins bearing a C-terminal EH, or
eps15 homology, domain. The EH domain has been found at the N-terminus of a small number of proteins, each of which has been implicated in endocytosis. RME-1 also contains a predicted P-loop sequence associated with ATP or GTP binding in many proteins, as well as a predicted coiled-coil domain. Homologs of RME-1 are found in the human and fly genome databases, but have not been studied functionally. No homologs of RME-1 are found in the complete yeast genome sequence. Sequencing of
rme-1 cDNAs and 5 RACE analysis identified four different
rme-1 mRNAs differing at their 5 ends, encoding four predicted RME-1 isoforms. Sequencing of our
rme-1 mutant alleles revealed mutations in highly conserved residues of the RME-1 region shared by all isoforms. Immunofluorescence with anti-RME-1 Abs shows that RME-1 is a cytoplasmic protein expressed in all cells, and that it is associated with the periphery of basolateral endosomes in the wild-type intestine.