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AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling

Nature Cell Biology volume 11, pages 1399–1410 (2009)Cite this article

Abstract

RME-1/EHD1 (receptor mediated endocytosis/Eps15 homology-domain containing 1) family proteins are key residents of the recycling endosome, which are required for endosome-to-plasma membrane transport in Caenorhabditis elegans and mammals. Recent studies suggest similarities between the RME-1/EHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. Here we show that endogenous C. elegans AMPH-1, the only C. elegans member of the Amphiphysin/BIN1 family of BAR (Bin1-Amphiphysin-Rvs161p/167p)-domain-containing proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 Asn-Pro-Phe(Asp/Glu) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1 (also known as Amphiphysin 2) in EHD1-regulated endocytic recycling. In vitro, we find that purified recombinant AMPH-1–RME-1 complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface.

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Figure 1: AMPH-1 physically interacts with RME-1.
Figure 2: AMPH-1 co-localizes with RME-1 and SDPN-1 on recycling endosomes.
Figure 3: C. elegans amph-1 mutants show abnormal trafficking of recycling cargo and morphologically abnormal recycling endosomes.
Figure 4: AMPH-1 function in endocytic recycling requires interaction with RME-1.
Figure 5: RNAi-mediated depletion of human Bin1 impairs transferrin recycling and disrupts mRme-1/EHD1-positive recycling endosome tubules.
Figure 6: Membrane binding and tubulation by AMPH-1 and RME-1 in vitro.
Figure 7: In vitro membrane binding and tubulation by AMPH-1 and RME-1 to PtdIns-containing liposomes.
Figure 8: Nucleotide effects on RME-1- and AMPH-1-mediated liposome tubulation.

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Acknowledgements

We thank P. McPherson, Z. Zhou, B. Kay, G. Prandergast and S. Mitani for important reagents. The monoclonal against RME-1 was made at Washington University School of Medicine and funded by Grant R24RR22234 (PI M. L. Nonet). We also thank C. Martin, P. Yurchenco, D. Winkelman, F. Matsumura, S. Yamashiro and S. H. deGregori for sharing their instruments, reagents and protocols; A. Shi for the construction and gift of the GFP–SDPN-1 strain; R. Patel and V. Starovoytov for expert assistance with electron microscopy protocols and Z. Pan for usage of the confocal microscopy facility; O. Daumke and T. Pucadyil for discussions and advice on liposome tubulation experiments; P. Schweinsberg for technical assistance. This work was supported by NIH Grants GM67237 to B.D.G and GM074876 to S.C., a NJCSCR Graduate Fellowship 06-2915-SCR-E-0, an Anne B. and James B. Leathem Fellowship, a McCallum Fund Fellowship Grant to S.P., an American Heart Association student fellowship to M.S. and a Rutgers Summer Undergraduate Research Fellowship (SURF) to K.P.

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Authors and Affiliations

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

    Saumya Pant, Kruti Patel & Barth D. Grant

  2. Department of Pathology and Laboratory Medicine, UMDNJ, Piscataway, 08854, NJ, USA

    Chavela M. Carr

  3. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, 68198, NE, USA

    Mahak Sharma & Steve Caplan

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Contributions

S.P. participated in experimental design, performed the C. elegans-related experiments and the biochemical and liposome experiments (Figs 1, 2, 3, 4, 6, 7, 8 and Supplementary Information, Figs S1–5 and S7) and wrote the manuscript. M.S. performed the mammalian cell experiments (Fig. 5 and Supplementary Information, Fig. S6) and helped to write those sections of the manuscript. K.P. contributed to developing some strains used in the study. S.C. designed the mammalian cell experiments. C.M.C. designed the experiments related to liposome biochemistry and trained S.P. to do those experiments. B.D.G. designed the experiments, trained S.P. in all C. elegans experiments and wrote the paper.

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Correspondence to Barth D. Grant.

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Pant, S., Sharma, M., Patel, K. et al. AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol 11, 1399–1410 (2009). https://doi.org/10.1038/ncb1986

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