The Hirsh lab

The Endocytosis Page

We study endocytosis, the process by which cells take up proteins. Endocytosis is used for taking up nutrients, processing antigens, recycling cell surface proteins and membranes, and regulating surface receptor density and activity. We have developed a new in vivo assay for studying receptor-mediated endocytosis in C. elegans. We have constructed transgenic worms that express a fusion of yolk protein with green fluorescent protein (ylk-GFP). Like normal yolk, ylk-GFP is synthesized in the intestine, secreted into the body cavity (pseudocoelom), and endocytosed by oocytes. Wildtype transgenic animals have bright green fluorescent oocytes and a pale body cavity. But animals defective in endocytosis have pale oocytes and a bright green fluorescent body cavity. These phenotypic differences form the basis for our ability to screen for mutants defective in endocytosis in order to identify new proteins that are important for the pathway. We have shown that C. elegans uses the same "classical" receptor mediated endocytosis pathway components as vertebrates by using RNA interference (RNAi) to block expression of C. elegans genes that are homologues of vertebrate endocytosis genes, e.g. clathrin heavy chain, alpha- and ß-adaptins, rab-5, -7, and -11, and dynamin. In each case, RNAi interferred with ylk-GFP uptake that resulted in diminished oocyte fluorescence and enhanced body cavity fluorescence. We used the assay to isolate endocytosis mutants that mapped to twelve different genes. We have studied three of the genes, rme -2, -8, and -1. rme-2 encodes the yolk receptor, which is a member of the LDL receptor family of proteins. It is expressed on the surface of the two most mature oocytes, and when ectopically expressed under the control of a muscle promoter, it causes yolk to bind to muscle cells. rme-8 encodes a large new member of the DNAJ domain family of proteins, and appears to function at an early step in the endocytosis pathway. rme-1 encodes a new member of the EH domain family of proteins, and functions in the recycling arm of the endocytosis pathway. RME-1 is expressed in all cells and localizes on what appear to be recycling endosomes and associated vesicles. Thus, we can now identify at will new components in the endocytosis pathway. Our tasks are to identify the products of other rme genes and sort out their molecular interactions using cell biology, biochemistry and genetics.

click here for more in formation on rme-8

Literature:

Greener, T., Grant, B., Zhang,Y., Wu, X., Greene, L.E., Hirsh, D., Eisenberg, E. (2001). Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo. Nature Cell Biol. 3, 215-219.

Lin, S.X., Grant, B., Hirsh, D., and Maxfield, F. (2001). Rme-1 Regulates the Distribution and Function of the Endocytic Recycling Compartment in Mammalian Cells. Nature Cell Biol. 3, 657-572.

Grant, B., Zhang, Y., Paupard, M.C., Lin, S.X., Hall, D.H., and Hirsh, D. (2001). Evidence that RME-1, a conserved C. elegans EH domain protein, functions in endocytic recycling. Nature Cell Biol. 3, 573-579.

Zhang, Y., Grant, B., and Hirsh, D. (2001). RME-8 a Conserved J-Domain Protein, Is Required in Endocytosis in C. elegans. Mol. Biol. Cell 12, 2011-2021.

Grant, B. and Hirsh, D. (1999). Receptor-mediated endocytosis in the C. elegans oocyte. Mol. Biol. Cell. 10, 4311-4326.

Xie, H. and Hirsh, D. (1998). In Vivo function of mutated spliced leader RNAs in Caenorhabditis elegans Proc. Natl. Acad. Sci. USA. 95, 4235-4240.

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