The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid from endosomes to the plasma membrane. We have previously shown that the EH-domain protein RME-1 is a critical regulator of endocytic recycling in worms and mammals. Here we identify the RAB-10 protein as a key regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the Caenorhabditis elegans intestine. rab-10 null mutant intestinal cells accumulate abnormally abundant RAB-5-positive early endosomes, some of which are enlarged by more than 10-fold. Conversely most RME-1-positive recycling endosomes are lost in rab-10 mutants. The abnormal early endosomes in rab-10 mutants accumulate basolaterally recycling transmembrane cargo molecules and basolaterally recycling fluid, consistent with a block in basolateral transport. These results indicate a role for RAB-10 in basolateral recycling upstream of RME-1. We found that a functional GFP-RAB-10 reporter protein is localized to endosomes and Golgi in wild-type intestinal cells consistent with a direct role for RAB-10 in this transport pathway.
INTRODUCTIONEndocytosis and endocytic trafficking controls the uptake and sorting of extracellular macromolecules as well as the components of the cell membrane itself, counterbalancing secretion and allowing a complex interplay between cells and their environment that is important for a myriad of cellular activities (Brodsky et al., 2001;Maxfield and McGraw, 2004). The steps involved in the uptake and trafficking of cargo within the endosomal system have been well described, but many of the components mediating these steps at the molecular level remain to be identified (Brodsky et al., 2001;Maxfield and McGraw, 2004). Many receptors and their associated ligands cluster into clathrincoated pits, whereas other types of cargo utilize clathrinindependent uptake mechanisms (Nichols, 2003;Gesbert et al., 2004). Plasma membrane invaginations pinch off into vesicles, uncoat, and then fuse with one another and with early endosomes. In early endosomes some ligand-receptor complexes dissociate because of the reduced pH of the endosomal lumen (Mukherjee et al., 1997). Many receptors then recycle to the plasma membrane (PM) either directly or indirectly via recycling endosomes (Mukherjee et al., 1997;Maxfield and McGraw, 2004). Many ligands do not recycle but instead are transported from early to late endosomes and eventually to lysosomes for degradation (Mukherjee et al., 1997). In polarized epithelial cells such as cultured Madin-Darby canine kidney (MDCK) cells, an additional layer of complexity in the endocytic pathway contributes to formation and/or maintenance of the specialized apical and basolateral domains (Nelson and Yeaman, 2001;Mostov et al., 2003;Hoekstra et al., 2004). Both the apical and basolateral membranes deliver cargo ...
