Study On The Mechanisms Of Action Of EHBP-1 Functional Domains

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 small GTPase RAB-10,is a critical regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the Caenorhabditis elegans intestine, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons.EHBP-1 (Ehbpl) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. EHBP-1 contains three distinct protein domains, including an N-terminal C2-like domain (NT-C2), central CH (Calponin Homology) domain, and C-terminal predicted coiled-coil (CC) domain. Our previous studies showed that the predicted CC domain of EHBP-1 binds to RAB-10(GTP),but the specific molecular regulation mechanism is not yet clear.We need to carry out the structure function studies to dissect the functional mechanism of EHBP-1.Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction,through the amino acid point mutation technology. Furthermore our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin micro filaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. Thus we propose a mechanism whereby RAB-10 stimulates EHBP-1 to tubulate endosomal membranes by linking them to the actin cytoskeleton. Our results suggest that local actin networks can stabilize tubular endosomal domains involved in the sorting and transport of recycling cargo.