Middle (e) and surface (f) of oocytes are shown
Middle (e) and surface (f) of oocytes are shown. plasma membrane to endosomes is usually defective, and small 100 nm endocytic vesicles accumulate just below the plasma membrane. These results Synpo suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin coated vesicles that is essential for the delivery of endocytic cargo to early endosomes. Introduction Clathrin-dependent endocytosis is the major route by which most receptor-ligand complexes are Dutasteride (Avodart) internalized. Clathrin and cargo molecules are put together into clathrin-coated pits around the plasma membrane (PM) together with adaptor proteins that link clathrin and transmembrane receptors, concluding in the formation of mature clathrin-coated vesicles (CCVs). CCVs are then actively uncoated and transported to early/sorting endosomes1. These processes are highly regulated but the precise molecular mechanisms controlling these steps are not fully understood. Small GTPases of the Rab family play pivotal functions in vesicular transport2. Rab5 in particular is known as a important regulator of the early endocytic pathway. Rab5 is found associated with the PM, CCVs, and most prominently with early endosomes3,4. Rab5 is usually thought to regulate homotypic fusion of early endosomes, microtubule-based motility of early endosomes, and the organization of subdomains within the early endosome limiting membrane, through its ordered recruitment of multiple effectors to the endosomal periphery5C8. studies also indicate that Rab5 regulates heterotypic fusion of CCVs with early endosomes, and in complex with GDI, Rab5 has been proposed to function in ligand sequestration during clathrin-coated pit formation9C11. To exert such diverse functions, the activity of Rab5 must be spatially and temporally controlled, probably at the level of nucleotide exchange by guanine nucleotide exchange factors (GEFs). Rabex-5 is the canonical Rab5 GEF and is essential for Rab5-dependent fusion of early endosomes mutants display endocytosis defects in oocytes and coelomocytes We isolated four alleles of in a previously reported genetic screen for mutants defective in oocyte endocytosis of YP170::GFP, a reporter for clathrin-dependent endocytosis in C. elegans16. In these mutants, YP170-uptake by oocytes was strongly reduced, resulting in accumulation of YP170::GFP in the body cavity and very little YP170::GFP in oocytes (RME phenotype; Fig.1 d). Seven alleles of were also identified in a different genetic screen for coelomocyte endocytosis mutants (Fig. 1e)17. As shown in Fig. 1f, mutants exhibit a high level accumulation Dutasteride (Avodart) of a fluid-phase endocytosis marker (GFP secreted by muscle mass) in the body cavity, indicating a severe defect in endocytosis by coelomocytes. These phenotypes in oocytes and coelomocytes suggest that is usually generally required for endocytosis in multiple tissues. Open in a separate windows Physique 1 mutant phenotypes and diagram of the predicted RME-6 protein. (aCd) YP170::GFP endocytosis by oocytes of adult hermaphrodites. In wild-type, YP170::GFP is usually efficiently endocytosed by oocytes (c). In the mutant, endocytosis of YP170::GFP by oocytes is usually greatly reduced and most YP170::GFP accumulates in the body cavity (d). Nomarski images Dutasteride (Avodart) of the same fields are also shown in a and b. Arrows indicate the position of oocytes. SP, spermatheca. (e and f) Coelomocyte endocytosis assay using Dutasteride (Avodart) GFP secreted from body-wall muscle mass cells. Secreted GFP is usually taken up by coelomocytes and accumulates in vesicles of wild-type coelomocytes (e). In strong accumulation of GFP in the body cavity was observed indicating poor endocytosis Dutasteride (Avodart) by coelomocytes (f). Some secreted GFP does accumulate within the coelomocytes of mutants, but the vesicles labeled by the internalized GFP are abnormally small (see Results). Cell boundaries of coelomocytes are layed out for clarity. Bar, 10 m. (g) Domain name structure of RME-6, showing the N-terminal RasGAP-related domain name and the Vps9 domain name at the C-terminus. Mutations recognized in each of the 11 alleles are also shown, including three missense mutations shown in strong. Nucleotide sequence changes in mutants: mutant worm and examined by immunoblotting using anti-RME-6 antibodies. Asterisk indicates a nonspecific band. encodes a conserved protein with RasGap-like and Vps9 domains We cloned the wild-type gene using standard methods and recognized sequence changes associated with each of the alleles (Fig. 1g and Supplemental physique S1; see materials and methods). The gene encodes a novel 1093 amino acid protein (Fig. 1g). The most notable feature of the predicted RME-6 sequence is usually a Vps9 domain name at the C-terminus, a motif found in the catalytic domains of Rab5 GEFs including human Rabex-5, RIN1, and yeast Vps9p12. In addition to the Vps9 domain name, RME-6 contains a RasGAP-related domain name in the N-terminal region18. We found predicted travel and mammalian homologues of RME-6 in genome databases, and the overall domain name structure is usually well conserved among them. The genome has a homologue of Rabex-5, the canonical Rab5 GEF,.