The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.
INTRODUCTIONReceptor-mediated endocytosis, an essential process in all eukaryotes, is required for general cellular functions, including uptake of nutrients (e.g., low-density lipoprotein [LDL] or transferrin) and recycling of membranes and membrane proteins (Mukherjee et al., 1997). Yolk uptake by growing oocytes is a dramatic example of the receptor-mediated endocytosis pathway in many species, including invertebrates such as the nematode Caenorhabditis elegans and vertebrates such as the chicken (Schneider, 1996). C. elegans yolk is secreted from its site of synthesis, the intestine, into the pseudoceolomic space (body cavity) and is ultimately taken up into vesicles within the growing oocytes (Kimble and Sharrock, 1983;Hall et al., 1999). Yolk transport in vertebrates such as the chicken follows a similar route, from liver to bloodstream to ovum (Schneider, 1996). Yolk is a lipoprotein particle composed of lipids and lipid-binding proteins called vitellogenins. Vitellogenins are among the most abundant proteins found in developing embryos (Sharrock, 1983). Lipids and proteins derived from yolk are thought to provide essential nutrients required to support the rapid development of the embryo. C. elegans vitellogenins YP170, YP115, and YP88 share sequence homology with vertebrate vitellogenins and with ApoB-100, a core component of mammalian LDL particles (Baker, 1988;Spieth et al., 1991). Endocytosis of yolk particles into membrane-bound vesicles of the oocyte is mediated by receptors of the LDL receptor superfamily in C. elegans (this work), insects, and vertebrates. Yolk and yolk receptor endocytic trafficking is thought to proceed through pathways very similar to those used by LDL in somatic cells Schneider, 1996).Clathrin-coated pit (CCP) and endosomal trafficking systems, such as those mediating LDL endocytosis, have been intensively studied since Roth and Porter (1964) first discovered coated-pits in the mosquito oocyte plasma membrane. Extensive ligand and receptor tracer studies have since produced a model describing general endocytic traffick...
