Permease Recycling and Ubiquitination Status Reveal a Particular Role for Bro1 in the Multivesicular Body Pathway
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m306953200
ISSN1083-351X
AutoresElina Nikko, Anne‐Marie Marini, Bruno André,
Tópico(s)Ubiquitin and proteasome pathways
ResumoUbiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation. We previously identified Bro1 as a protein essential to this down-regulation. In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis. Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles. Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi. This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function. Ubiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation. We previously identified Bro1 as a protein essential to this down-regulation. In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis. Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles. Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi. This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function. The machineries responsible for endocytosis and degradation of plasma membrane proteins are crucial to normal regulation of cell surface-associated functions such as transport, signaling, and adhesion (1.D'Hondt K. Heese-Peck A. Riezman H. Annu. Rev. Genet. 2000; 34: 255-295Crossref PubMed Scopus (104) Google Scholar, 2.Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar). The proteins are first internalized from the cell surface into small endocytic vesicles, a step that generally requires prior ubiquitination of cargo proteins at the plasma membrane. The endocytosed proteins then move through early and late endosomes to finally reach the lysosomal/vacuolar lumen where they are degraded. The last steps of this endocytic pathway, from endosome to vacuolar lumen, are shared with a subset of biosynthetic membrane proteins derived from the late secretory pathway (3.Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar). Namely, the delivery of both endocytic and biosynthetic membrane proteins to the lysosomal/vacuolar lumen requires their prior sorting into the multivesicular body (MVB) 1The abbreviations used are: MVBmultivesicular bodyVpsvacuolar protein sortingESCRTendosomal sorting complex required for transportt-SNAREtarget-soluble N-ethylmaleimide attachment protein receptorTGNtrans-Golgi networkCPScarboxypeptidase SGFPgreen fluorescent protein.1The abbreviations used are: MVBmultivesicular bodyVpsvacuolar protein sortingESCRTendosomal sorting complex required for transportt-SNAREtarget-soluble N-ethylmaleimide attachment protein receptorTGNtrans-Golgi networkCPScarboxypeptidase SGFPgreen fluorescent protein. pathway (4.Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (433) Google Scholar, 5.Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). MVBs arise from endosomes, as the protein-carrying endosomal membrane invaginates and buds into the endosome, forming internal vesicles. The proteins sorted to these MVB vesicles are ultimately delivered to the lumen of the lysosome/vacuole when the MVB fuses with this organelle. Besides its role in the early steps of endocytosis, ubiquitin is also important for MVB sorting, as entry of several proteins into MVB vesicles depends on their prior ubiquitination (6.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 7.Reggiori F. Pelham H.R. EMBO J. 2001; 20: 5176-5186Crossref PubMed Scopus (271) Google Scholar, 8.Urbanowski J.L. Piper R.C. Traffic. 2001; 2: 622-630Crossref PubMed Scopus (118) Google Scholar). In yeast mutants defective in the class E Vps factors, proteins normally targeted to the vacuolar lumen accumulate in an aberrant late endosome, referred to as the class E compartment, as well as at the limiting membrane of the vacuole (5.Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 9.Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (671) Google Scholar). Several of these Vps factors are organized in three distinct endosome-associated protein complexes. The ESCRT-I complex participates in recognition of ubiquitinated proteins that are targeted into the MVB pathway (6.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). ESCRT-II and -III function downstream from ESCRT-I, sorting proteins into MVB internal vesicles (10.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 11.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). The MVB pathway involves several other factors as follows: the Vps27-Hse1 complex to recruit the ESCRT-I complex and to recognize ubiquitinated MVB cargo proteins (12.Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar, 13.Shih S.C. Katzmann D.J. Schnell J.D. Sutanto M. Emr S.D. Hicke L. Nat. Cell Biol. 2002; 4: 389-393Crossref PubMed Scopus (358) Google Scholar); the AAA-type ATPase Vps4 to dissociate the ESCRT complexes from the endosomal membrane; and the ubiquitin hydrolase Doa4, recruited by ESCRTIII, to deubiquitinate proteins prior to their sorting into MVB vesicles (6.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 14.Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (613) Google Scholar, 15.Amerik A.Y. Nowak J. Swaminathan S. Hochstrasser M. Mol. Biol. Cell. 2000; 11: 3365-3380Crossref PubMed Scopus (261) Google Scholar). The MVB sorting machinery appears well conserved, because most of the yeast class E Vps proteins have mammalian homologues whose functions are probably similar to those of their yeast counterparts (16.Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar).We use the yeast general amino acid permease Gap1 as a model to gain further insight into the process of regulated trafficking of membrane proteins. Gap1 is suitable for this purpose, as its membrane trafficking is tightly controlled by the nature of the nitrogen source. Under conditions of poor nitrogen supply (proline or urea), Gap1 is abundantly synthesized and accumulates in a highly active and stable form at the plasma membrane. Upon addition of a preferential nitrogen source like ammonium (NH4+), the GAP1 gene is repressed, and the permease present at the cell surface is rapidly poly-ubiquitinated on two lysine residues in the N-terminal tail (Lys-9 and Lys-16). This ubiquitination triggers Gap1 endocytosis followed by sorting to the vacuole/lysosome lumen for degradation (17.Grenson M. Eur. J. Biochem. 1983; 133: 135-139Crossref PubMed Scopus (88) Google Scholar, 18.Jauniaux J.C. Grenson M. Eur. J. Biochem. 1990; 190: 39-44Crossref PubMed Scopus (231) Google Scholar, 19.Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. André B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (296) Google Scholar, 20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar, 21.Springael J.Y. Galan J.M. Haguenauer-Tsapis R. André B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar, 22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Ubiquitination of Gap1 at the cell surface is dependent on the ubiquitin ligase Rsp5/Npi1, the Bul proteins, and the Doa4/Npi2 ubiquitin hydrolase, the latter being required to maintain a normal level of free ubiquitin (19.Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. André B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (296) Google Scholar, 20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar, 22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 23.Swaminathan S. Amerik A.Y. Hochstrasser M. Mol. Biol. Cell. 1999; 10: 2583-2594Crossref PubMed Scopus (239) Google Scholar). The nitrogen source controls Gap1 trafficking also in the late secretory pathway (24.Roberg K.J. Rowley N. Kaiser C.A. J. Cell Biol. 1997; 137: 1469-1482Crossref PubMed Scopus (161) Google Scholar). Although newly synthesized Gap1 is sorted from the Golgi to the plasma membrane in proline- or urea-grown cells, it is directed to the vacuole, bypassing the cell surface, when glutamate (24.Roberg K.J. Rowley N. Kaiser C.A. J. Cell Biol. 1997; 137: 1469-1482Crossref PubMed Scopus (161) Google Scholar), ammonium (22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), or other amino acids (25.Chen E.J. Kaiser C.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14837-14842Crossref PubMed Scopus (76) Google Scholar) are present. The same direct sorting of Gap1 to the vacuole occurs on proline or urea medium in cells lacking a functional Npr1 (26.De Craene J.O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), a protein kinase inhibited under favored nitrogen supply (17.Grenson M. Eur. J. Biochem. 1983; 133: 135-139Crossref PubMed Scopus (88) Google Scholar, 27.Vandenbol M. Jauniaux J.C. Grenson M. Mol. Gen. Genet. 1990; 222: 393-399Crossref PubMed Scopus (81) Google Scholar, 28.Schmidt A. Beck T. Koller A. Kunz J. Hall M.N. EMBO J. 1998; 17: 6924-6931Crossref PubMed Scopus (261) Google Scholar). Like endocytosis, this direct vacuolar sorting of Gap1 requires ubiquitination (22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) or even polyubiquitination (29.Helliwell S.B. Losko S. Kaiser C.A. J. Cell Biol. 2001; 153: 649-662Crossref PubMed Scopus (237) Google Scholar) of the permease.Down-regulation of Gap1 also requires the NPI3 gene: in npi3 mutant cells, the permease stays mainly at the plasma membrane after ammonium addition. The cells also show an apparent defect in Gap1 ubiquitination (30.Springael J.Y. Nikko E. André B. Marini A.M. FEBS Lett. 2002; 517: 103-109Crossref PubMed Scopus (50) Google Scholar). The npi3 mutant was initially isolated together with npi1 and npi2 strains in a screen for mutations restoring high Gap1 activity in the npr1 mutant (17.Grenson M. Eur. J. Biochem. 1983; 133: 135-139Crossref PubMed Scopus (88) Google Scholar). Accordingly, Npi3 is also required for direct sorting of Gap1 to the vacuole that occurs in the absence of Npr1, indicating that the Npi3 function is not limited to Gap1 endocytosis (30.Springael J.Y. Nikko E. André B. Marini A.M. FEBS Lett. 2002; 517: 103-109Crossref PubMed Scopus (50) Google Scholar). Cloning of NPI3 revealed that it is identical to BRO1, a gene originally described to interact genetically with components of the Pkc1-mitogen-activated protein kinase pathway (31.Nickas M.E. Yaffe M.P. Mol. Cell. Biol. 1996; 16: 2585-2593Crossref PubMed Scopus (65) Google Scholar). BRO1/NPI3 has also been identified as ASI6; the asi6 mutation restores amino acid uptake in cells defective in a signaling pathway that induces transcription of several amino acid permease genes (32.Forsberg H. Hammar M. Andreasson C. Moliner A. Ljungdahl P.O. Genetics. 2001; 158: 973-988PubMed Google Scholar). Bro1/Npi3/Asi6 has also been isolated in two separate screens for factors required for vacuolar protein sorting (5.Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 33.Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (723) Google Scholar, 34.Bonangelino C.J. Chavez E.M. Bonifacino J.S. Mol. Biol. Cell. 2002; 13: 2486-2501Crossref PubMed Scopus (265) Google Scholar) and was named Vps31. For simplicity, the protein will hereafter be referred to as Bro1. In a recent study, Bro1 was shown to be a soluble class E Vps factor associating with endosomes and required for the MVB sorting of the vacuolar hydrolase carboxypeptidase S (CPS) (35.Odorizzi G. Katzmann D.J. Babst M. Audhya A. Emr S.D. J. Cell Sci. 2003; 116: 1893-1903Crossref PubMed Scopus (173) Google Scholar). Bro1 is conserved throughout the eukaryotic kingdom, and its homologues include mouse AIP1/Alix, a cytosolic protein involved in apoptosis but whose function has remained unclear (36.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (210) Google Scholar, 37.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar).Here we report that Bro1 is not essential for ubiquitination or for endocytosis of Gap1. Instead, the protein intervenes at a late step of MVB sorting, likely after the ESCRT-III core components and before or in conjunction with the Doa4-deubiquitinating enzyme. In contrast to other class E Vps factors of the ESCRT-I, -II, and -III complexes, a deficiency in Bro1 leads to recycling of the permease back to the cell surface via the trans-Golgi. Interestingly, this recycling requires normal endosometo-vacuole transport. Furthermore, we provide evidence that recycling of Gap1 is accompanied by deubiquitination of the permease.EXPERIMENTAL PROCEDURESStrains, Plasmids, and Media—The Saccharomyces cerevisiae strains used in this study (Table I) are isogenic with Σ1278b (38.Béchet J. Greenson M. Wiame J.M. Eur. J. Biochem. 1970; 12: 31-39Crossref PubMed Scopus (191) Google Scholar). The gene deletions and excision of the KanMX4 cassette were performed as described elsewhere (22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The primer sequences are available upon request. Cells were transformed as described previously (22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The plasmids used in this study are listed in Table I. Plasmid pJOD10 is identical to pCJ025 (26.De Craene J.O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Cells were grown in minimal buffered medium (pH 6.1) (39.Jacobs P. Jauniaux J.C. Grenson M. J. Mol. Biol. 1980; 139: 691-704Crossref PubMed Scopus (112) Google Scholar) with 3% glucose as the carbon source except where indicated otherwise. Nitrogen sources were added as indicated to the following final concentrations: (NH4)2SO4, 10 mm; proline, 0.1%.Table IStrains and plasmids used in this studyStrainGenotypeRef.EK008gap1Δ ura3Laboratory collectionEN121gap1Δ end3Δ ura3This studyEN053gap1Δ vps23Δ ura3This studyEN057gap1Δ vps36Δ ura3This studyEN054gap1Δ snf7Δ ura3This studyEN050gap1Δ vps4Δ ura3This studyEN076gap1Δ bro1Δ ura3This studyCJ013gap1Δ ypt6Δ ura3Jacob and AndreaC. Jacob and B. Andre, unpublished data36216bgap1Δ bro1Δ ypt6Δ ura3This study23344cura3Laboratory collection27092abro1Δ ura330.Springael J.Y. Nikko E. André B. Marini A.M. FEBS Lett. 2002; 517: 103-109Crossref PubMed Scopus (50) Google Scholar36222dgap1Δ bro1Δ end3Δ ura3This studyEN123gap1Δ pep12Δ ura3This studyEN124gap1Δ bro1Δ pep12Δ ura3This studyEN080gap1Δ bro1Δ vps23Δ ura3This studyEN082gap1Δ bro1Δ snf7Δ ura3This studyEN064vam3Δ ura3This studyEN083bro1Δ vam3Δ ura3This study36210abro1Δ pep4Δ ura3This study36233bgap1Δ vam3Δ ura3This study36234agap1Δ bro1Δ vam3Δ ura3This studyPlasmid YCpFL38CEN-ARS URA377.Bonneaud N. Ozier-Kalogeropoulos O. Li G.Y. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (500) Google Scholar YCpGap1YCpFL38 GAP122.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar YCpGap1K9K16YCpFL38 GAP1K9K1622.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar p416 GAL1CEN-ARS URA378.Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (793) Google Scholar pJOD10p416 GAL1 GAP1-GFP26.De Craene J.O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholara C. Jacob and B. Andre, unpublished data Open table in a new tab Permease Assays—Cells carrying the gap1Δ mutation were transformed with the YCpGap1 or YCpGap1K9K16 plasmid. Gap1 permease activity was determined by measuring the initial rate of incorporation of 14C-labeled citrulline (20 μm), a specific substrate of the permease (40.Grenson M. Mousset M. Wiame J.M. Bechet J. Biochim. Biophys. Acta. 1966; 127: 325-338Crossref PubMed Scopus (271) Google Scholar). Gap1 was inactivated by adding (NH4)2SO4 (10 mm) to the culture. The permease activity was determined in each strain at least three times.Yeast Cell Extracts and Immunoblotting—Cells bearing the gap1Δ mutation were transformed with plasmid YCpGap1 or YCpGap1K9K16. Crude extracts (19.Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. André B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (296) Google Scholar) and membrane-enriched preparations (20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar) were obtained as described previously. Gap1 immunoblotting was performed as described (22.Soetens O. De Craene J.O. André B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Extract preparation and Gap1 immunoblotting were performed a minimum of three times for each strain.Fluorescence Microscopy—Cells transformed with the pJOD10 plasmid were grown in minimal buffered medium (pH 6.1) with 3% galactose and 0.3% glucose as carbon sources and 0.1% proline as the nitrogen source. Glucose was added to the final concentration of 3% 2.5 h before starting the experiment in order to stop Gap1-GFP synthesis. Gap1-GFP internalization was induced by adding (NH4)2SO4 (10 mm)to the culture. To visualize vacuoles, cells were incubated in medium containing 40 μm FM4-64 (Molecular Probes, Eugene, OR), shaken for 15 min at 30 °C, transferred to fresh medium, and chased for ∼1 h. Cells were viewed with a Nikon Eclipse E600 microscope equipped with appropriate fluorescence light filter sets. Images were captured with a Nikon DXM1200 digital camera and processed using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). All the Gap1 location experiments were performed at least three times.RESULTSGap1 Is Sorted into the MVB Pathway—To date, a number of yeast proteins have been shown to reach the vacuolar lumen via the MVB pathway (e.g. CPS, Ste2, and Fur4) (5.Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 41.Marchal C. Dupré S. Urban-Grimal D. J. Cell Sci. 2002; 115: 217-226PubMed Google Scholar). The sorting of these proteins into intra-endosomal vesicles necessitates the interplay of several class E Vps factors, many of which are organized in complexes (6.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 10.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 11.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). In class E vps mutants, membrane proteins normally transiting through the MVB pathway mainly accumulate in an aberrant late endosome referred to as the class E compartment and/or are missorted to the vacuole limiting membrane. We thus sought to determine the fate of Gap1 after ammonium addition to various mutants lacking one of the following class E Vps factors: Vps23, Vps36, Snf7/Vps32 (components of the ESCRT-I, -II, and -III complexes, respectively), or Vps4 (the AAA-type ATPase required for ESCRT complex dissociation from the endosomal membrane) (6.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 10.Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 11.Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar).The presence of Gap1 at the cell surface is reflected by permease activity that is easily monitored by 14C-labeled citrulline uptake assay. In accordance with previous studies (19.Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. André B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (296) Google Scholar, 20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar), we observed high Gap1 activity in proline-grown wild type cells; this activity decreases rapidly after addition of 20 mmNH4+ (Fig. 1A). In end3Δ cells defective in endocytosis (42.Raths S. Rohrer J. Crausaz F. Riezman H. J. Cell Biol. 1993; 120: 55-65Crossref PubMed Scopus (319) Google Scholar), however, the permease activity remained substantially high (Fig. 1A), as shown previously with thermosensitive act1 mutant cells (20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar). The internalization process was also studied by using a green fluorescent protein-tagged version of the permease (Gap1-GFP). The Gap1-GFP protein is correctly directed to the cell surface where it is functional and largely sensitive toNH4+-triggered down-regulation. Internalization of the chimeric protein is somewhat delayed, however, compared with native Gap1, so that 2 h afterNH4+ addition some permease activity is still measured. 2J.-O. De Craene, E. Nikko, and B. André, unpublished observations. In proline-grown wild type cells (t = 0 min), Gap1-GFP was present mainly at the plasma membrane. Addition ofNH4+ induced progressive removal of the permease from the cell surface and its targeting to the vacuolar lumen (t = 120 min) (Fig. 1B). Immunodetection of Gap1 in crude extracts indeed showed degradation of the permease in response toNH4+ (Fig. 1C) (20.Springael J.Y. André B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (184) Google Scholar).Addition ofNH4+ resulted in a significant loss of Gap1 activity also in the various class E vpsΔ cells (Fig. 1A), but the internalized permease did not reach the lumen of the vacuole. Instead,NH4+ addition led to a progressive shift of Gap1-GFP in all vpsΔ cells from the cell surface to compartments next to the vacuole, with weak fluorescence on the vacuole limiting membrane, as indicated by FM4-64 staining (Fig. 1B). The structures adjacent to the vacuole likely represent the class E compartment (43.Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1127) Google Scholar), and the internalized Gap1 thus appears to accumulate mainly on the membrane limiting this compartment instead of being sorted to the vacuolar lumen. Accordingly, immunoblots showed that the Gap1 protein was not degraded in the various vpsΔ mutants (Fig. 1C). In all vpsΔ cells, addition ofNH4+ also induced the appearance of several additional bands above the main Gap1 signal. These were sustained during the whole experiment. As discussed below, these upper bands correspond to ubiquitin-conjugated forms of Gap1.Hence, Gap1 is normally internalized but not targeted to the vacuolar lumen in mutants lacking components of the ESCRT complexes. Instead, the permease accumulates in a limited number of punctate structures likely corresponding to the class E compartment, whereas a fraction of the protein also reaches the limiting vacuolar membrane.Gap1 Is Internalized but Recycled Back to the Cell Surface in bro1Δ Cells—Bro1 was recently described as a class E Vps factor associated with endosomal membranes and required for MVB sorting of CPS (35.Odorizzi G. Katzmann D.J. Babst M. Audhya A. Emr S.D. J. Cell Sci. 2003; 116: 1893-1903Crossref PubMed Scopus (173) Google Scholar). Interestingly, we independently identified Bro1 as a factor essential to normal ubiquitination and subsequent down-regulation of Gap1 and other permeases including the uracil permease Fur4 (30.Springael J.Y. Nikko E. André B. Marini A.M. FEBS Lett. 2002; 517: 103-109Crossref PubMed Scopus (50) Google Scholar). After ammonium addition to bro1Δ mutant cells, Gap1 remains highly active and is not degraded (Fig. 2, A and C) (30.Springael J.Y. Nikko E. André B. Marini A.M. FEBS Lett. 2002; 517: 103-109Crossref PubMed Scopus (50) Google Scholar). Consistently, most of the Gap1-GFP remained at the cell surface even 2 h afterNH4+ addition (Fig. 2B). However, some fluorescence was clearly found also on the vacuole limiting membrane, as indicated by staining with the lipophilic dye FM4-64. This suggests that in bro1Δ cells a fraction of the Gap1 is internalized but that its sorting to the vacuolar lumen is impaired. This phenotype is markedly different from that displayed by the above-mentioned class E Vps-factor mutants, where Gap1 is internalized and accumulates largely in the class E compartment, whereas only a fraction of the permease reaches the limiting membrane of the vacuole.Fig. 2A recycling defect restores ubiquitin-dependent internalization of Gap1 in bro1Δ cells.A, Gap1 activity was measured in wild type (Wt) (▪, EK008), bro1Δ (□, EN076), ypt6Δ (•, CJ013), and bro1Δ ypt6Δ cells (○, 36216b) transformed with plasmid YCpGap1 and in bro1Δ ypt6Δ cells transformed with the YCpGap1K9K16 plasmid (▴, 36216b) before (t = 0 min) and several times afterNH4+ addition, as in Fig. 1. B, location of Gap1-GFP before (t = 0 min) and after (t = 120 min)NH4+ addition to wild type (EK008), bro1Δ (EN076), and bro1Δ ypt6Δ cells (36216b) transformed with plasmid pJOD10. Note the absence of fluorescence in the vacuolar lumen both in bro1▵ and bro1▵ ypt6▵ cells. Cells were labeled with FM4-64 to stain the vacuolar membrane and endosomal compartments. C, Western analysis of Gap1 in total protein extracts prepared before (t = 0 min) and at several times after addition ofNH4+. Strains were as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)That Gap1 resides largely at the plasma membrane in bro1Δ cells afterNH4+ addition suggests that Bro1 might play a role in some early steps of endocytosis (e.g. in Gap1 ubiquitination) in addition to MVB sorting. Alternatively, Gap1 might be efficiently internalized in bro1Δ cells but not correctly sorted to the vacuole. As a consequence, the permease would largely recycle back to the cell surface, a smaller fraction being missorted to the limiting vacuolar membrane (Fig. 2B). To test the latter hypothesis, we isolated bro1Δ strains containing additional mutations that impair transport from the endosomes to the trans-Golgi network (TGN), a step required for recycling of Chs3 (chitin synthase III) and Snc1 (v-SNARE) from the endocytic pathway back to the plasma membrane (44.Holthuis J.C. Nichols B.J. Pelham H.R. Mol. Biol. Cell. 1998; 9: 3383-3397Crossref PubMed Scopus (95) Google Scholar, 45.Lewis M.J. Nichols B.J. Prescianotto-Baschong C. Riezman H. Pelham H.R. Mol. Biol. Cell. 2000; 11: 23-38Crossref PubMed Scopus (277) Google Scholar). Endosome-to-Golgi transport depends on several factors, such as the Golgi-associated GTPase Ypt6, the VFT complex (Vps51-Vps52-Vps53-Vps54), and the late Golgi/endosomal SNARE proteins Tlg1 and Tlg2 (44.Holthuis J.C. Nichols B.J. Pelham H.R. Mol. Biol. Cell. 1998; 9: 3383-3397Crossref PubMed Scopus (95) Google Scholar, 46.Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 47.Conibear E. Stevens T.H. Mol. Biol
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