Endocytosis is required in cells for diverse functions such as nutrient uptake, synaptic vesicle recycling, plasma membrane recycling and receptor internalization. We have isolated a number of C. elegans endocytic mutants using a vitellogenin::GFP assay (Grant and Hirsh, 1999). Here we describe the molecular analysis and phenotypic characterization of
rme-8 (receptor-mediated endocytosis mutant 8).
rme-8 encodes a predicted protein containing 2279 amino acids with a central DnaJ domain. DnaJ domains are known to stimulate the ATPase activity of chaperone proteins of the
hsc70 family. Together they function to modulate the folding and unfolding of proteins. rme- 8 homologues are found in humans, Drosophila and Arabidopsis, but not in yeast. This sequence conservation suggests RME-8 may define a conserved step in endocytosis in multicellular organisms.
rme-8 is required for endocytosis in most worm cells.
rme-8(
b1023) is defective in both receptor-mediated endocytosis in oocytes and fluid-phase endocytosis in coelomocytes. This allele is also temperature sensitive for lethality, reflecting a broad requirement for
rme-8 activity in other cells. Temperature-shift experiments indicate a requirement for
rme-8 activity during all developmental stages. Consistent with its role in endocytosis, RME-8 is localized to intracellular endocytic structures. Using a reporter consisting of an RME-8::GFP chimera that rescues the
rme-8 mutant, we found that
rme-8 is expressed in many cells. RME-8::GFP localizes to small intracellular punctate structures, except in coelomocytes, where it localizes to the periphery of large vesicles beneath the plasma membrane. These large RME-8::GFP vesicles are pre-lysosomal endosomes. By antibody staining, we found that RME-8 localizes to cortical punctate structures in the oocyte which are not early endosomes. In summary, we have identified a new conserved endocytic gene
rme-8. We showed that
rme-8 is essential and is required for endocytosis in many cells. We propose that
rme-8 functions in an intermediate intracellular trafficking step prior to lysosome formation.