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Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit

Nature Cell Biology volume 7, pages 559–569 (2005)Cite this article

Abstract

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)–RME-6 fusion protein primarily localizes to clathrin-coated 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 110-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.

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Figure 1: rme-6 mutant phenotypes and the predicted RME-6 protein.
Figure 2: RME-6 physically interacts with RAB-5S33N.
Figure 3: rme-6 and rabx-5 function in membrane recruitment of GFP–RAB-5, and in animal viability.
Figure 4: An early stage of endocytosis slows down in rme-6 mutant coelomocytes.
Figure 5: rme-6 accumulates chains of small yolk-receptor-positive vesicles.
Figure 6: Expression and subcellular localization of GFP–RME-6 in worm tissues.
Figure 7: GFP–RME-6 colocalizes strongly with clathrin-coated pit markers.
Figure 8: Physical and functional association of RME-6 and coated-pit proteins.

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Acknowledgements

We thank H. Fares, Y. Zhang, D. Hirsh and G. Seydoux for important reagents; R. Y. Tsien for mRFP plasmids; S. Mitani for the rabx-5 knockout strain; S. Yamashiro and F. Matsumura for help with mammalian cell culture; and I. Greenwald and H. Fares for critical reading of this manuscript. We also thank P. Schweinsberg, L. Pedraza and W. Przylecki for expert technical assistance. B.D.G. is especially grateful to D. Hirsh for his strong support during the early phases of this work. M.S. and K.S. were supported by JSPS Postdoctoral Fellowship for Research Abroad and Bioarchitect Research Projects of RIKEN, respectively. This work was supported by a NIH Grant GM67237 and MOD Grant 5-FY02-252 to B.D.G. B.D.G. also received support from the Chicago Community Trust Searle Scholars Program. W.L. was supported by a Grant NSC 91-2320-B-182-034 of National Science Council of Taiwan.

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Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, 08854, NJ, USA

    Miyuki Sato, Ken Sato, Paul Fonarev & Barth D. Grant

  2. Department of Anatomy, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan, 333, R.O.C.

    Chih-Jen Huang & Willisa Liou

  3. Molecular Membrane Biology Laboratory, RIKEN,, Wako, 351-0198, Saitama, Japan

    Ken Sato

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Sato, M., Sato, K., Fonarev, P. et al. Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit. Nat Cell Biol 7, 559–569 (2005). https://doi.org/10.1038/ncb1261

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