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. 2003 Jun;164(2):469–477. doi: 10.1093/genetics/164.2.469

Opposite roles of the F-box protein Rcy1p and the GTPase-activating protein Gyp2p during recycling of internalized proteins in yeast.

Céline Lafourcade 1, Jean-Marc Galan 1, Matthias Peter 1
PMCID: PMC1462592  PMID: 12807768

Abstract

The F-box protein Rcy1p is part of a non-SCF (Skp1p-cullin-F-box protein) complex involved in recycling of internalized material. Like rcy1Delta, cells lacking the Rab-GTPase Ypt6p or its heterodimeric GEFs Rgp1p and Ric1p are unable to recycle the v-SNARE Snc1p. Here we provide genetic evidence suggesting that Rcy1p is a positive regulator of Ypt6p. Deletion of the GAP Gyp2p restores recycling in rcy1Delta, while overexpression of an active form of Ypt6p partially suppresses the recycling defect of rcy1Delta cells. Conversely, overexpression of Gyp2p in wild-type cells interferes with recycling of GFP-Snc1p, and the cells accumulate membrane structures as evidenced by electron microscopy. Gyp2p-GFP is distributed throughout the cytoplasm and accumulates in punctate structures, which concentrate in an actin-dependent manner at sites of polarized growth. Taken together, our results suggest that the F-box protein Rcy1p may activate the Ypt6p GTPase module during recycling.

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Selected References

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  1. Albert S., Gallwitz D. Msb4p, a protein involved in Cdc42p-dependent organization of the actin cytoskeleton, is a Ypt/Rab-specific GAP. Biol Chem. 2000 May-Jun;381(5-6):453–456. doi: 10.1515/BC.2000.059. [DOI] [PubMed] [Google Scholar]
  2. Albert S., Gallwitz D. Two new members of a family of Ypt/Rab GTPase activating proteins. Promiscuity of substrate recognition. J Biol Chem. 1999 Nov 19;274(47):33186–33189. doi: 10.1074/jbc.274.47.33186. [DOI] [PubMed] [Google Scholar]
  3. Bai C., Sen P., Hofmann K., Ma L., Goebl M., Harper J. W., Elledge S. J. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996 Jul 26;86(2):263–274. doi: 10.1016/s0092-8674(00)80098-7. [DOI] [PubMed] [Google Scholar]
  4. Bi E., Chiavetta J. B., Chen H., Chen G. C., Chan C. S., Pringle J. R. Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast. Mol Biol Cell. 2000 Feb;11(2):773–793. doi: 10.1091/mbc.11.2.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burns N., Grimwade B., Ross-Macdonald P. B., Choi E. Y., Finberg K., Roeder G. S., Snyder M. Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. Genes Dev. 1994 May 1;8(9):1087–1105. doi: 10.1101/gad.8.9.1087. [DOI] [PubMed] [Google Scholar]
  6. Chen L., Davis N. G. Recycling of the yeast a-factor receptor. J Cell Biol. 2000 Oct 30;151(3):731–738. doi: 10.1083/jcb.151.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Linyi, Davis Nicholas G. Ubiquitin-independent entry into the yeast recycling pathway. Traffic. 2002 Feb;3(2):110–123. doi: 10.1034/j.1600-0854.2002.030204.x. [DOI] [PubMed] [Google Scholar]
  8. Di Rienzo A., Peterson A. C., Freimer N. B. Amplification with arbitrary primers. Methods Mol Biol. 1996;54:123–129. doi: 10.1385/0-89603-313-9:123. [DOI] [PubMed] [Google Scholar]
  9. 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. 1998 Feb 6;273(6):3253–3256. doi: 10.1074/jbc.273.6.3253. [DOI] [PubMed] [Google Scholar]
  10. Du L. L., Novick P. Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p. Mol Biol Cell. 2001 May;12(5):1215–1226. doi: 10.1091/mbc.12.5.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galan J. M., Peter M. Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9124–9129. doi: 10.1073/pnas.96.16.9124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galan J. M., Wiederkehr A., Seol J. H., Haguenauer-Tsapis R., Deshaies R. J., Riezman H., Peter M. Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast. Mol Cell Biol. 2001 May;21(9):3105–3117. doi: 10.1128/MCB.21.9.3105-3117.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holthuis J. C., Nichols B. J., Pelham H. R. The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast. Mol Biol Cell. 1998 Dec;9(12):3383–3397. doi: 10.1091/mbc.9.12.3383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lewis M. J., Nichols B. J., Prescianotto-Baschong C., Riezman H., Pelham H. R. Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Mol Biol Cell. 2000 Jan;11(1):23–38. doi: 10.1091/mbc.11.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Longtine M. S., McKenzie A., 3rd, Demarini D. J., Shah N. G., Wach A., Brachat A., Philippsen P., Pringle J. R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998 Jul;14(10):953–961. doi: 10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  16. Siniossoglou S., Peak-Chew S. Y., Pelham H. R. Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p. EMBO J. 2000 Sep 15;19(18):4885–4894. doi: 10.1093/emboj/19.18.4885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Siniossoglou S., Pelham H. R. An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes. EMBO J. 2001 Nov 1;20(21):5991–5998. doi: 10.1093/emboj/20.21.5991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Strom M., Vollmer P., Tan T. J., Gallwitz D. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature. 1993 Feb 25;361(6414):736–739. doi: 10.1038/361736a0. [DOI] [PubMed] [Google Scholar]
  19. Wiederkehr A., Avaro S., Prescianotto-Baschong C., Haguenauer-Tsapis R., Riezman H. The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae. J Cell Biol. 2000 Apr 17;149(2):397–410. doi: 10.1083/jcb.149.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wysocki R., Roganti T., Van Dyck E., de Kerchove D'Exaerde A., Foury F. Disruption and basic phenotypic analysis of 18 novel genes from the yeast Saccharomyces cerevisiae. Yeast. 1999 Jan 30;15(2):165–171. doi: 10.1002/(SICI)1097-0061(19990130)15:2<165::AID-YEA351>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
  21. Zhang F. L., Casey P. J. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996;65:241–269. doi: 10.1146/annurev.bi.65.070196.001325. [DOI] [PubMed] [Google Scholar]
  22. Ziman M., Chuang J. S., Tsung M., Hamamoto S., Schekman R. Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae. Mol Biol Cell. 1998 Jun;9(6):1565–1576. doi: 10.1091/mbc.9.6.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. von Bartheld C. S., Wang X., Butowt R. Anterograde axonal transport, transcytosis, and recycling of neurotrophic factors: the concept of trophic currencies in neural networks. Mol Neurobiol. 2001 Aug-Dec;24(1-3):1–28. doi: 10.1385/MN:24:1-3:001. [DOI] [PubMed] [Google Scholar]

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