The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells. INTRODUCTIONLysosomes are ubiquitous membrane-bound organelles that function as major degradative sites within eukaryotic cells (Tappel, 1969). Lysosomes contain an assortment of aciddependent hydrolases that function in the breakdown of proteins, lipids, nucleic acids, and oligosaccharides. Lysosomes receive exogenous material through the endocytic pathway and are characterized as being the terminal compartment of the endocytic pathway. Lysosomes also receive material via the secretory pathway and directly from the cytoplasm (Kornfeld and Mellman, 1989;Mullins and Bonifacino, 2001;Luzio et al., 2003). Lysosomes function in diverse and important cellular processes including cell surface receptor turnover, destruction of pathogens, antigen processing, digestion, starvation responses, tissue remodeling, ion storage, autophagy, and plasma membrane repair.The yeast vacuole shares several characteristics with the lysosomes of higher animals. Genetic screens have led to the identification of Ͼ150 genes necessary for the transport and sorting of newly synthesized proteins to the yeast vacuole (Jones, 1977;Bankaitis et al., 1986;Rothman and Stevens, 1986;Bonangelino et al., 2002). These genes control two pathways of Golgi-to-vacuole transport, the carboxypeptidase Y (CPY) and alkaline phosphatase (ALP) sorting pathways (Burd et al., 1998;Conibear and Stevens, 1998;Mullins and Bonifacino, 2001). Proteins trafficked via the CPY pathway transit an endosomal prevacuolar compartment en route to the vacuole. The ALP pathway mediates transport to the vacuole independent of the prevacuolar compartment.Many of the genes involved in transport to the yeast vacuole have homologues in higher animals (Lemmon and Traub, 2000;Mullins and Bonifacino, 2001;Bonangelino et al., 2002). For example, the HOPS complex proteins (Vps11p, Vps16p, Vps18p, and Vps33p) regulate membrane fusion events necessary for lysosomal delivery within yeast (Rieder and Emr, 1997;Peterson and Emr, 2001), Drosophila melanogaster (Sevrioukov et al., 1999;Sriram et al., 2003), and mammalian (Poupon et al., 2003;Richardson et al., 2004) endosomal systems. Similarly, the proteins compos...

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.

Using C. elegans genetics we identified a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains RasGAP-like and Vsp9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to C. elegans Rab5 in the GDP-bound conformation, and rme-6 mutants display phenotypes that indicate low Rab5 activity. Furthermore, rme-6 interacts genetically with the worm homologue of the known Rab5 exchange factor Rabex-5. However, unlike other Rab5 associated proteins, a rescuing GFP::RME-6 fusion protein primarily localizes to clathrincoated pits, physically interacts with α-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants transport from the plasma membrane to endosomes is defective, and small 100 nm endocytic vesicles accumulate just below the plasma membrane. These results 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.

Abbreviations used in the paper: CCV, clathrin-coated vesicle; GEF, guanine nucleotide exchange factor; PP2A, protein phosphatase 2A; PtdIns(4,5)P 2 , phosphoinositide 4,5-bisphosphate; Tfn, transferrin; TfnR, tranferrin receptor.The online version of this article contains supplemental material.