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Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP

Nature Structural & Molecular Biology volume 12, pages 38–45 (2005)Cite this article

Abstract

Transport protein particle (TRAPP) is a large multiprotein complex involved in endoplasmic reticulum–to–Golgi and intra-Golgi traffic. TRAPP specifically and persistently resides on Golgi membranes. Neither the mechanism of the subcellular localization nor the function of any of the individual TRAPP components is known. Here, the crystal structure of mouse Bet3p (bet3), a conserved TRAPP component, reveals a dimeric structure with hydrophobic channels. The channel entrances are located on a putative membrane-interacting surface that is distinctively flat, wide and decorated with positively charged residues. Charge-inversion mutations on the flat surface of the highly conserved yeast Bet3p led to conditional lethality, incorrect localization and membrane trafficking defects. A channel-blocking mutation led to similar defects. These data delineate a molecular mechanism of Golgi-specific targeting and anchoring of Bet3p involving the charged surface and insertion of a Golgi-specific hydrophobic moiety into the channels. This essential subunit could then direct other TRAPP components to the Golgi.

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Figure 1: Overall structure, hydrophobic channel and S-acylation of bet3.
Figure 2: Unusually flat surface of bet3.
Figure 3: Secondary structure assignment and sequence alignment.
Figure 4: Phenotype of wild-type and mutant yeast Bet3p.
Figure 5: Model for Golgi-specific targeting and localization of TRAPP.

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Protein Data Bank

References

  1. Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S. & Novick, P. Protein complexes in transport vesicle targeting. Trends Cell Biol. 10, 251–255 (2000).

    Article  CAS  Google Scholar 

  2. Whyte, J.R. & Munro, S. Vesicle tethering complexes in membrane traffic. J. Cell Sci. 115, 2627–2637 (2002).

    CAS  Google Scholar 

  3. Lee, M.C.S., Miller, E.A., Goldberg, J., Orci, L. & Schekman, R. Bi-directional protein transport between the ER and Golgi. Annu. Rev. Cell Dev. Biol. 20, 87–123 (2004).

    Article  CAS  Google Scholar 

  4. Sacher, M. et al. TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J. 17, 2494–503 (1998).

    Article  CAS  Google Scholar 

  5. Sacher, M. et al. TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport. Mol. Cell 7, 433–442 (2001).

    Article  CAS  Google Scholar 

  6. Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    Article  CAS  Google Scholar 

  7. Sacher, M., Barrowman, J., Schieltz, D., Yates, J.R. 3rd & Ferro-Novick, S. Identification and characterization of five new subunits of TRAPP. Eur. J. Cell Biol. 79, 71–80 (2000).

    Article  CAS  Google Scholar 

  8. Wang, W., Sacher, M. & Ferro-Novick, S. TRAPP stimulates guanine nucleotide exchange on Ypt1p. J. Cell Biol. 151, 289–296 (2000).

    Article  CAS  Google Scholar 

  9. Gedeon, A.K. et al. Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda. Nat. Genet. 22, 400–404 (1999).

    Article  CAS  Google Scholar 

  10. Barrowman, J., Sacher, M. & Ferro-Novick, S. TRAPP stably associates with the Golgi and is required for vesicle docking. EMBO J. 19, 862–869 (2000).

    Article  CAS  Google Scholar 

  11. Rossi, G., Kolstad, K., Stone, S., Palluault, F. & Ferro-Novick, S. BET3 encodes a novel hydrophilic protein that acts in conjunction with yeast SNAREs. Mol. Biol. Cell 6, 1769–1780 (1995).

    Article  CAS  Google Scholar 

  12. Cole, N.B., Ellenberg, J., Song, J., DiEuliis, D. & Lippincott-Schwartz, J. Retrograde transport of Golgi-localized proteins to the ER. J. Cell Biol. 140, 1–15 (1998).

    Article  CAS  Google Scholar 

  13. Munro, S. Organelle identity and the targeting of peripheral membrane proteins. Curr. Opin. Cell Biol. 14, 506–514 (2002).

    Article  CAS  Google Scholar 

  14. Lemmon, M.A. Phosphoinositide recognition domains. Traffic 4, 201–213 (2003).

    Article  CAS  Google Scholar 

  15. Bijlmakers, M.J. & Marsh, M. The on-off story of protein palmitoylation. Trends Cell Biol. 13, 32–42 (2003).

    Article  CAS  Google Scholar 

  16. Liu, J., Hughes, T.E. & Sessa, W.C. The first 35 amino acids and fatty acylation sites determine the molecular targeting of endothelial nitric oxide synthase into the Golgi region of cells: a green fluorescent protein study. J. Cell. Biol. 137, 1525–1535 (1997).

    Article  CAS  Google Scholar 

  17. Shenoy-Scaria, A.M., Dietzen, D.J., Kwong, J., Link, D.C. & Lublin, D.M. Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae. J. Cell Biol. 126, 353–363 (1994).

    Article  CAS  Google Scholar 

  18. Murray, D. et al. Electrostatics and the membrane association of Src: theory and experiment. Biochemistry 37, 2145–2159 (1998).

    Article  CAS  Google Scholar 

  19. Brennwald, P. & Novick, P. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4. Nature 362, 560–563 (1993).

    Article  CAS  Google Scholar 

  20. Zhou, W. & Resh, M.D. Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. J. Virol. 70, 8540–8548 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. McCabe, J.B. & Berthiaume, L.G. Functional roles for fatty acylated amino-terminal domains in subcellular localization. Mol. Biol. Cell 10, 3771–3786 (1999).

    Article  CAS  Google Scholar 

  22. Resh, M.D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1–16 (1999).

    Article  CAS  Google Scholar 

  23. Pfeffer, S.R. Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol. 11, 487–491 (2001).

    Article  CAS  Google Scholar 

  24. Sivars, U., Aivazian, D. & Pfeffer, S.R. Yip3 catalyses the dissociation of endosomal Rab–GDI complexes. Nature 425, 856–859 (2003).

    Article  CAS  Google Scholar 

  25. Uetz, P. et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

    Article  CAS  Google Scholar 

  26. Ito, T. et al. Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins. Proc. Natl. Acad. Sci. USA 97, 1143–1147 (2000).

    Article  CAS  Google Scholar 

  27. Schroder, S., Schimmoller, F., Singer-Kruger, B. & Riezman, H. The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in α-COP. J. Cell Biol. 131, 895–912 (1995).

    Article  CAS  Google Scholar 

  28. Otte, S. et al. Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. J. Cell Biol. 152, 503–518 (2001).

    Article  CAS  Google Scholar 

  29. Stevens, T., Esmon, B. & Schekman, R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30, 439–448 (1982).

    Article  CAS  Google Scholar 

  30. Tholey, A., Pipkorn, R., Bossemeyer, D., Kinzel, V. & Reed, J. Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase. Biochemistry 40, 225–231 (2001).

    Article  CAS  Google Scholar 

  31. Finger, F.P., Hughes, T.E. & Novick, P. Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, 559–571. (1998).

    Article  CAS  Google Scholar 

  32. Rak, A. et al. Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase. Science 302, 646–650 (2003).

    Article  CAS  Google Scholar 

  33. Jang, S.B. et al. Crystal structure of SEDL and its implications for a genetic disease spondyloepiphyseal dysplasia tarda. J. Biol. Chem. 277, 49863–49869 (2002).

    Article  CAS  Google Scholar 

  34. Dietrich, L.E., Gurezka, R., Veit, M. & Ungermann, C. The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion. EMBO J. 23, 45–53 (2004).

    Article  CAS  Google Scholar 

  35. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  36. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000).

    Article  CAS  Google Scholar 

  37. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  38. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  39. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  40. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  41. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to M. Cygler (Biotechnology Research Institute) for invaluable input and discussions on this work, J. Wagner for technical assistance, and T. Stevens (University of Oregon) and S. Ferro-Novick (Yale University) for providing strains and reagents. This study made use of beamline 6B at Pohang Accelerator Laboratory. This work was supported by Creative Research Initiatives of the Korean Ministry of Science & Technology and by the Réseau Protéomique de Montréal Proteomics Network. Y.-G.K. was supported by the Brain Korea 21 Project.

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Authors and Affiliations

  1. Department of Life Science and Division of Molecular and Life Sciences, Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, 790-784, Kyungbuk, Korea

    Yeon-Gil Kim & Byung-Ha Oh

  2. Department of Life Science and Division of Molecular and Life Sciences, Center for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang, 790-784, Kyungbuk, Korea

    Eun Ju Sohn & Inhwan Hwang

  3. Division of Molecular Life Sciences and College of Pharmacy, Center for Cell Signaling Research, Ewha Womans University, Seoul, 120-750, Korea

    Jawon Seo & Kong-Joo Lee

  4. Pohang Accelerator Laboratory, Pohang, 790-784, Kyungbuk, Korea

    Heung-Soo Lee

  5. Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, H4P 2R2, Quebec, Canada

    Malcolm Whiteway

  6. Montreal Proteomics Network, 6100 Royalmount Avenue, Montreal, H4P 2R2, Quebec, Canada

    Michael Sacher

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Supplementary information

Supplementary Fig. 1

Enlargement of the electron density for myristoyl-Cys68. (PDF 47 kb)

Supplementary Fig. 2

CD spectra of yeast Bet3p proteins. (PDF 49 kb)

Supplementary Fig. 3

ESI mass spectrum of bet3(8–172) produced in E. coli. (PDF 43 kb)

Supplementary Methods

Supplementary Methods (PDF 13 kb)

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Kim, YG., Sohn, E., Seo, J. et al. Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP. Nat Struct Mol Biol 12, 38–45 (2005). https://doi.org/10.1038/nsmb871

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