Intrinsic tethering activity of endosomal Rab proteins

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Kornmann, B. et al. An ER-mitochondria tethering complex revealed by a synthetic biology screen. Science 325, 477–481 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Grosshans, B.L., Ortiz, D. & Novick, P. Rabs and their effectors: achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. USA 103, 11821–11827 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Barr, F.A. Rab GTPase function in Golgi trafficking. Semin. Cell Dev. Biol. 20, 780–783 (2009).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Singer-Kruger, B. et al. Role of three Rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J. Cell Biol. 125, 283–298 (1994).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Horazdovsky, B.F., Busch, G.R. & Emr, S.D. VPS21 encodes a Rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J. 13, 1297–1309 (1994).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Gerrard, S.R., Bryant, N.J. & Stevens, T.H. VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment. Mol. Biol. Cell 11, 613–626 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Gorvel, J.P., Chavrier, P., Zerial, M. & Gruenberg, J. Rab5 controls early endosome fusion in vitro. Cell 64, 915–925 (1991).

    CAS  Article  Google Scholar 

  8. 8

    Bucci, C. et al. Co-operative regulation of endocytosis by three Rab5 isoforms. FEBS Lett. 366, 65–71 (1995).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Barbieri, M.A. et al. Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion. J. Biol. Chem. 273, 25850–25855 (1998).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Simonsen, A. et al. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394, 494–498 (1998).

    CAS  Article  Google Scholar 

  11. 11

    Christoforidis, S., McBride, H.M., Burgoyne, R.D. & Zerial, M. The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621–625 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Christoforidis, S. et al. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat. Cell Biol. 1, 249–252 (1999).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Lawe, D.C. et al. Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1. J. Biol. Chem. 277, 8611–8617 (2002).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Davie, E.W. A brief historical review of the waterfall/cascade of blood coagulation. J. Biol. Chem. 278, 50819–50832 (2003).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Antonny, B., Madden, D., Hamamoto, S., Orci, L. & Schekman, R. Dynamics of the COPII coat with GTP and stable analogues. Nat. Cell Biol. 3, 531–537 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Wollert, T. & Hurley, J.H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864–869 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Drin, G., Morello, V., Casella, J.F., Gounon, P. & Antonny, B. Asymmetric tethering of flat and curved lipid membranes by a golgin. Science 320, 670–673 (2008).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Hickey, C.M. & Wickner, W. HOPS initiates vacuole docking by tethering membranes prior to trans-SNARE complex assembly. Mol. Biol. Cell 21, 2297–2305 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Hickey, C.M., Stroupe, C. & Wickner, W. The major role of the Rab Ypt7p in vacuole fusion is supporting HOPS membrane association. J. Biol. Chem. 284, 16118–16125 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Stroupe, C., Hickey, C.M., Mima, J., Burfeind, A.S. & Wickner, W. Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components. Proc. Natl. Acad. Sci. USA 106, 17626–17633 (2009).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Peterson, M.R., Burd, C.G. & Emr, S.D. Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome. Curr. Biol. 9, 159–162 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Tall, G.G., Hama, H., DeWald, D.B. & Horazdovsky, B.F. The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting. Mol. Biol. Cell 10, 1873–1889 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Horazdovsky, B.F., Cowles, C.R., Mustol, P., Holmes, M. & Emr, S.D. A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization. J. Biol. Chem. 271, 33607–33615 (1996).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Peplowska, K., Markgraf, D.F., Ostrowicz, C.W., Bange, G. & Ungermann, C. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev. Cell 12, 739–750 (2007).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Markgraf, D.F. et al. The CORVET subunit Vps8 cooperates with the Rab5 homolog Vps21 to induce clustering of late endosomal compartments. Mol. Biol. Cell 20, 5276–5289 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Plemel, R.L. et al. Subunit organization and Rab interactions of Vps-C protein complexes that control endolysosomal membrane traffic. Mol. Biol. Cell 22, 1353–1363 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Gureasko, J. et al. Membrane-dependent signal integration by the Ras activator Son of sevenless. Nat. Struct. Mol. Biol. 15, 452–461 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. A 411, 177–184 (1987).

    CAS  Article  Google Scholar 

  30. 30

    Wang, L., Seeley, E.S., Wickner, W. & Merz, A.J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle. Cell 108, 357–369 (2002).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Patel, S.S. & Rexach, M.F. Discovering novel interactions at the nuclear pore complex using bead halo: a rapid method for detecting molecular interactions of high and low affinity at equilibrium. Mol. Cell. Proteomics 7, 121–131 (2008).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Braun, P. et al. An experimentally derived confidence score for binary protein-protein interactions. Nat. Methods 6, 91–97 (2009).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Chen, Y.C., Rajagopala, S.V., Stellberger, T. & Uetz, P. Exhaustive benchmarking of the yeast two-hybrid system. Nat. Methods 7, 667–668 author reply 668 (2010).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Brett, C.L. et al. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase. J. Cell Biol. 182, 1141–1151 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37

    Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Daitoku, H., Isida, J., Fujiwara, K., Nakajima, T. & Fukamizu, A. Dimerization of small GTPase Rab5. Int. J. Mol. Med. 8, 397–404 (2001).

    CAS  PubMed  Google Scholar 

  39. 39

    Pawelec, A., Arsic, J. & Kolling, R. Mapping of Vps21 and HOPS binding sites in Vps8 and effect of binding site mutants on endocytic trafficking. Eukaryot. Cell 9, 602–610 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Hama, H., Tall, G.G. & Horazdovsky, B.F. Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport. J. Biol. Chem. 274, 15284–15291 (1999).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Buvelot Frei, S. et al. Bioinformatic and comparative localization of Rab proteins reveals functional insights into the uncharacterized GTPases Ypt10p and Ypt11p. Mol. Cell. Biol. 26, 7299–7317 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  42. 42

    Du, L.L., Collins, R.N. & Novick, P.J. Identification of a Sec4p GTPase-activating protein (GAP) as a novel member of a Rab GAP family. J. Biol. Chem. 273, 3253–3256 (1998).

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Pan, X., Eathiraj, S., Munson, M. & Lambright, D.G. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442, 303–306 (2006).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Lee, M.C. et al. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 122, 605–617 (2005).

    CAS  Article  Google Scholar 

  45. 45

    Pucadyil, T.J. & Schmid, S.L. Conserved functions of membrane active GTPases in coated vesicle formation. Science 325, 1217–1220 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Wittmann, J.G. & Rudolph, M.G. Crystal structure of Rab9 complexed to GDP reveals a dimer with an active conformation of switch II. FEBS Lett. 568, 23–29 (2004).

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Pasqualato, S. et al. The structural GDP/GTP cycle of Rab11 reveals a novel interface involved in the dynamics of recycling endosomes. J. Biol. Chem. 279, 11480–11488 (2004).

    CAS  Article  PubMed  Google Scholar 

  48. 48

    Scapin, S.M. et al. The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform. J. Struct. Biol. 154, 260–268 (2006).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Beck, R. et al. Membrane curvature induced by Arf1-GTP is essential for vesicle formation. Proc. Natl. Acad. Sci. USA 105, 11731–11736 (2008).

    CAS  Article  PubMed  Google Scholar 

  50. 50

    Chapman, E.R. How does synaptotagmin trigger neurotransmitter release? Annu. Rev. Biochem. 77, 615–641 (2008).

    CAS  Article  Google Scholar 

  51. 51

    Li, F. et al. Energetics and dynamics of SNAREpin folding across lipid bilayers. Nat. Struct. Mol. Biol. 14, 890–896 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52

    Schwartz, M.L. & Merz, A.J. Capture and release of partially zipped trans-SNARE complexes on intact organelles. J. Cell Biol. 185, 535–549 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Nielsen, E. et al. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151, 601–612 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54

    Furgason, M.L. et al. The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p. Proc. Natl. Acad. Sci. USA 106, 14303–14308 (2009).

    CAS  Article  Google Scholar 

  55. 55

    Sinka, R., Gillingham, A.K., Kondylis, V. & Munro, S. Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins. J. Cell Biol. 183, 607–615 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56

    Hayes, G.L. et al. Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi. Mol. Biol. Cell 20, 209–217 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Angers, C.G. & Merz, A.J. New links between vesicle coats and Rab-mediated vesicle targeting. Semin. Cell Dev. Biol. 22, 18–26 (2011).

    CAS  Article  PubMed  Google Scholar 

  58. 58

    Ohya, T. et al. Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes. Nature 459, 1091–1097 (2009).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Alexey J Merz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–4 and Supplementary Methods (PDF 2106 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lo, S., 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

Download citation

Further reading