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Intrinsic tethering activity of endosomal Rab proteins

Nature Structural & Molecular Biology volume 19, pages 40–47 (2012)Cite this article

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Abstract

Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion.

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Figure 1: GTP-bound Vps21 tethers liposomes.
Figure 2: Vps21 surface density and tethering activity.
Figure 3: Vps21 interactions in trans are required for efficient tethering.
Figure 4: The Vps21 C-terminal linker is not required for tethering.
Figure 5: Vps21-GTP interacts with known effectors and with itself in yeast two-hybrid assays.
Figure 6: Vps21 interacts with Ypt53 and Ypt10 to drive GTP-dependent heterotypic tethering.
Figure 7: Regulation and reversibility of Vps21-mediated liposome tethering.
Figure 8: Model for Rab-Rab driven tethering in endosome docking and fusion.

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Acknowledgements

We thank P. Brennwald and J. Taraska for helpful discussion and D. Baker and R. Koga for assistance with multiangle light scattering experiments. High-throughput screening and support of M.V. was through the Yeast Resource Center (US National Institutes of Health (NIH) P41 RR11823). S.L. was supported in part by University of Washington Nanotechnology Integrative Graduate Education and Research Traineeship award (US National Science Foundation DGE-0504573). We thank the Murdock Charitable Trust and the Washington Research Foundation for generous support of our electron cryomicroscopy laboratory. T.G. was a Howard Hughes Medical Institute early career scientist and S.F. is a Howard Hughes Medical Institute investigator. This work was supported by NIH grant GM077349 and research scholar grant 10-026-01-CSM from the American Cancer Society.

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  1. Christopher L Brett & Tamir Gonen

    Present address: Present addresses: Department of Biology, Concordia University, Montreal, Quebec, Canada (C.L.B.); and Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA (T.G.).,

Authors and Affiliations

  1. Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington, USA

    Sheng-Ying Lo, Christopher L Brett, Rachael L Plemel, Tamir Gonen & Alexey J Merz

  2. Department of Chemistry, University of Washington, Seattle, Washington, USA

    Sheng-Ying Lo

  3. Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington, USA

    Marissa Vignali & Stanley Fields

  4. Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington, USA

    Stanley Fields & Tamir Gonen

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Contributions

S.Y.L. and A.J.M. conceived the project. S.Y.L. developed and validated the QLS-based tethering system; expressed, purified and characterized proteins; prepared liposomes and carried out and interpreted all QLS tethering experiments. C.L.B. and A.J.M. conceived and C.L.B. and S.Y.L. implemented the fluorescence microscopy-based tethering assays. T.G. did the E M. S.F. and M.V. developed the high-throughput yeast two-hybrid technology, and R.L.P. and M.V. executed and interpreted yeast two-hybrid screens and assays. S.Y.L. and A.J.M. wrote the paper.

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Correspondence to Alexey J Merz.

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Lo, SY., Brett, C., Plemel, R. et al. Intrinsic tethering activity of endosomal Rab proteins. Nat Struct Mol Biol 19, 40–47 (2012). https://doi.org/10.1038/nsmb.2162

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