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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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.
The authors declare no competing financial interests.
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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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