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• W2080442977 abstract "In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH4+, a preferential source of nitrogen, is added to cells growing on proline or urea,i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1K9K16) remains fully stable at the plasma membrane after NH4+ addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1K9 and Gap1K16 mutants, in which a single lysine is mutated, are down-regulated in response to NH4+ although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939–43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Δ mutant, neosynthesized Gap1K9K16 is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Δ mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking,i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi. In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH4+, a preferential source of nitrogen, is added to cells growing on proline or urea,i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1K9K16) remains fully stable at the plasma membrane after NH4+ addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1K9 and Gap1K16 mutants, in which a single lysine is mutated, are down-regulated in response to NH4+ although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939–43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Δ mutant, neosynthesized Gap1K9K16 is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Δ mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking,i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi. general amino acid permease ubiquitin in which lysine residues 29, 48, and 63 are replaced by arginine vacuolar protein sorting ubiquitin-conjugating enzymes epidermal growth factor receptor homologous to E6-AP C terminus Ubiquitin is a 76-amino acid protein, which, in all eukaryotes, undergoes conjugation to a multitude of proteins. Although ubiquitination generally serves as a recognition signal for degradation by the proteasome (1Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar, 2Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (706) Google Scholar), studies in yeast have shown that ubiquitination of plasma membrane proteins results in their endocytosis followed by vacuolar degradation (3Hicke L. FASEB J. 1997; 11: 1215-1226Crossref PubMed Scopus (229) Google Scholar). Proteins subject to this mechanism include the G-protein-coupled mating pheromone receptors Ste2 (4Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar) and Ste3 (5Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar) and several transporters: the ABC proteins Ste6 (6Kölling R. Hollenberg C.P. EMBO J. 1994; 13: 3261-3271Crossref PubMed Scopus (271) Google Scholar) and Pdr5 (7Egner R. Kuchler K. FEBS Lett. 1996; 378: 177-181Crossref PubMed Scopus (102) Google Scholar), the uracil permease Fur4 (8Galan J. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (323) Google Scholar), the amino acid permease Gap1 (9Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar, 10Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar), the tryptophan permease Tat2 (11Beck T. Schmidt A. Hall M.N. J. Cell Biol. 1999; 146: 1227-1238Crossref PubMed Scopus (251) Google Scholar), the galactose permease Gal2 (12Horak J. Wolf D.H. J. Bacteriol. 1997; 179: 1541-1549Crossref PubMed Google Scholar), and the zinc transporter Zrt1 (13Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar). Ubiquitination of most of these proteins has been shown to involve the ubiquitin-conjugating enzymes (E2) encoded by the UBC1–4 genes and an HECT-type ubiquitin ligase (E3) encoded by the essential NPI1/RSP5 gene (14Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar). Ubiquitin has been shown to contain an endocytosis signal in the form of two surface patches surrounding two critical residues (Phe4 and Ile44) (15Shih S.C. Sloper-Mould K.E. Hicke L. EMBO J. 2000; 19: 187-198Crossref PubMed Scopus (245) Google Scholar). However, the protein components of the endocytosis machinery involved in ubiquitin recognition remain unknown. It also remains undetermined as to whether ubiquitin also plays a role in the late steps of endocytosis and whether plasma membrane proteins undergo successive cycles of ubiquitination-de-ubiquitination during transit to the vacuole. Here we have investigated the role of ubiquitin in the internal trafficking of the general amino acid permease (Gap1),1 which is tightly regulated by nitrogen. On proline or urea medium, i.e.conditions of poor nitrogen supply, the GAP1 gene is transcribed to high levels (16Jauniaux J.C. Grenson M. Eur. J. Biochem. 1990; 190: 39-44Crossref PubMed Scopus (234) Google Scholar), and the synthesized Gap1 permease accumulates at the plasma membrane in an active and stable form (17Grenson M. Eur. J. Biochem. 1983; 133: 141-144Crossref PubMed Scopus (77) Google Scholar,18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Upon the addition of NH4+ (a preferential source of nitrogen), Gap1 is internalized by endocytosis and targeted to the vacuole for degradation. Ubiquitination of Gap1 is essential to this NH4+-induced down-regulation (9Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar, 10Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). In the npi1 mutant, which displays an abnormally low level of the HECT-type ubiquitin ligase Npi1/Rsp5, or the npi2 mutant lacking the Npi2/Doa4 de-ubiquitinating enzyme, Gap1 is not ubiquitinated and stays at the plasma membrane after NH4+ addition (10Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar,19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar). Furthermore, as shown for the uracil permease Fur4 (8Galan J. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (323) Google Scholar), Gap1 is poly-ubiquitinated, the ubiquitin moieties being attached to the lysine 63 of the preceding ubiquitin (19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar) (henceforth called the lysine 63-linked poly-ubiquitin chain). Gap1 poly-ubiquitination is required for down-regulation of the permease at a maximal rate (19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar). The fate of newly synthesized Gap1 in the late secretory pathway is also under nitrogen control. On proline or urea medium, neosynthesized Gap1 is delivered to the plasma membrane, but in a medium containing glutamate (20Roberg K.J. Rowley N. Kaiser C.A. J. Cell Biol. 1997; 137: 1469-1482Crossref PubMed Scopus (161) Google Scholar) or NH4+ (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) as the sole nitrogen source, Gap1 is directly sorted from the Golgi to the vacuole without passing via the cell surface. A similar situation has been observed on proline medium with cells lacking Npr1, a protein kinase controlling both cell-surface and internal Gap1 (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and apparently inactivated by phosphorylation when good nitrogen sources are available (21Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In this paper we show that Gap1 is ubiquitinated on two lysine residues in its extreme N terminus, at positions 9 and 16. Using the Gap1K9K16 variant in which both lysine residues are mutated, we show that ubiquitination of Gap1 is required not only for down-regulation of the protein pre-accumulated at the cell surface but also for direct sorting of the protein from the late secretory pathway to the vacuole, as occurs in an npr1Δ mutant. We further show that ubiquitination and degradation of both cell-surface and internal Gap1 requires Bul1 and Bul2, two proteins interacting with the Npi1/Rsp5 ubiquitin ligase. As this paper was being reviewed, it was reported by others (22Helliwell S.B. Losko S. Kaiser C.A. J. Cell Biol. 2001; 153: 649-662Crossref PubMed Scopus (237) Google Scholar) that sorting of Gap1 to the vacuole requires its poly-ubiquitination and that the specific role of Bul1 and Bul2 is to specify this modification (see “Discussion”). All Saccharomyces cerevisiae strains used in this study (see Table I) are isogenic with Σ1278b (23Béchet J. Grenson M. Wiame J.M. Eur. J. Biochem. 1970; 12: 31-39Crossref PubMed Scopus (192) Google Scholar). Cells were grown in minimal buffered medium (24Jacobs 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 when mentioned otherwise. In steady-state experiments, proline (10 mm) was the sole nitrogen source. In experiments of Gap1 neosynthesis, cells were grown exponentially on glutamine (5 mm) or NH4+ (100 mm) and transferred to proline medium to relieve GAP1 repression. In ubiquitin overexpression experiments, cells were grown on glutamine medium and transferred to preheated YNB (yeast nitrogen base without NH4+ or amino acids; Difco) medium containing 10 mm proline, 3% glucose, and 0.1 mmCuSO4 to induce expression of ubiquitin. The 2 μ-based multi-copy plasmid YEp96 contains a synthetic yeast ubiquitine gene under the control of the copper-inducible CUP1 promoter (25Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4606-4610Crossref PubMed Scopus (197) Google Scholar). The plasmids encoding the ubiquitin in which lysine residue 63 (UbK63R), or lysine residues 29, 48, and 63 (UbRRR) are replaced by arginine are derived from YEp96 (26Arnason T. Ellison M.J. Mol. Cell. Biol. 1994; 14: 7876-7883Crossref PubMed Scopus (196) Google Scholar). Centromeric plasmid YCpGAP1 (27Hein C. Andre B. Mol. Microbiol. 1997; 24: 607-616Crossref PubMed Scopus (67) Google Scholar) is based on the YCpFL38 plasmid (28Bonneaud N. Ozier-Kalogeropoulos O. Li G.Y. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (501) Google Scholar). Plasmid YCpFL39 was used to complement trp1 auxotrophy (28Bonneaud N. Ozier-Kalogeropoulos O. Li G.Y. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (501) Google Scholar).Table IStrains used in this studyStrainGenotypeReference23344cMATα ura3Laboratory collection30788aMATα npr1∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)27038aMATa npi1 ura3(54Grenson M. Eur. J. Biochem. 1983; 133: 135-139Crossref PubMed Scopus (88) Google Scholar)30788dMATα npi1 npr1∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33191bMATa KanMX2-GAL-GAP1 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33192cMATa KanMX2-GAL-GAP1 npr1∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33308cMATa KanMX2-GAL-GAP1 pep12∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33307aMATa KanMX2-GAL-GAP1 npr1∷KanMX2 pep12∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33201bMATa KanMX2-GAL-GAP1 npi1 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)33191aMATa KanMX2-GAL-GAP1 npi1 npr1∷KanMX2 ura3(18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar)27061bMATa trp1 ura3(19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar)27071bMATa npi2 trp1 ura3(19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar)36012cMATa npr1∷KanMX2 trp1 ura3This study36005bMATa npi2 npr1∷KanMX2 trp1 ura3This study30629aMATα gap1∷KanMX2Laboratory collectionOS21-1MATa gap1∷KanMX2 npi2 trp1 ura3This studyJA369MATa gap1∷KanMX2 npr1-1 ura3This studyJA364MATα bul1∷KanMX4 ura3This studyJA379MATα bul2∷KanMX4 ura3This studyOS27-1MATα bul1∷KanMX4 bul2∷KanMX4 ura3This studyJA410MATα bul1∷KanMX4 bul2∷KanMX4 npr1∷KanMX4 ura3This study Open table in a new tab Site-directed mutagenesis of GAP1was performed using the Quick Change Site-directed Mutagenesis Kit (Stratagene) on plasmid YCpGAP1 as recommended by the supplier. The primers used for each construct are described in TableII. Each construct was checked entirely by sequencing.Table IIOligonucleotides used in this studyPrimerPurposePrimer sequenceDEL1NPR1deletion5′-TAG TAC GGA TTA GTC AGT GGC GTA CCT AGT GGC AAC AAT CGC GGC CGC CAG CTG AAG CTT CGT ACG C-3′DEL2NPR1deletion5′-AGT AGA TTA TGA ACA GGA GGT CAA TCT ATT TAG GCT TCT ATA GCG GCC GCA TAG GCC ACT AGT GGA TCT G-3′OR5K9K16 mutagenesis5′-CG TAC GAG AGG AAT AAT CCA GAT AAT CTG AGA CAC AAT GG-3′OR6K9K16 mutagenesis5′-CC ATT GTG TCT CAG ATT ATC TGG ATT ATT CCT CTC GTA CG-3′OR7K51K56K60K63 mutagenesis5′-GT TCA GGG TCC AGA TGG CAA GAC TTT AGA GAT TCT TTC AGA AGG GTA AGA CCT ATT GAA G-3′OR8K51K56K60K63 mutagenesis5′-C TTC AAT AGG TCT TAC CCT TCT GAA AGA ATC TCT AAA GTC TTG CCA TCT GGA CCC TGA AC-3′OR11K9 mutagenesis5′-CT TCG TAC GAG AGG AAT AAT CCA GAT AAT C-3′OR12K9 mutagenesis5′-G ATT ATC TGG ATT ATT CCT CTC GTA CGA AG-3′OR13K16 mutagenesis5′-CCA GAT AAT CTG AGA CAC AAT GGT ATT ACC-3′OR14K16 mutagenesis5′-GGT AAT ACC ATT GTG TCT CAG ATT ATC TGG-3′D5-BUL1BUL1 deletion5′-G AGA CTG TTC GTG TGT GTC AAC AGG TAT ATC GTA CGC TAA GCG GCC GCC AGC TGA AGC TT-3′D3-BUL1BUL1 deletion5′-A TCT ATA AGA AAA GTA ACG AGA ATT TTT TCT AAT GTT TTT GCG GCC GCA TAG GCC ACT AG-3′D5-BUL2BUL2 deletion5′-G CAG ATT TGA GAT ATA TTC TGG GGA ACA AAA GAA GTA TTA GCG GCC GCC AGC TGA AGC TT-3′D3-BUL2BUL2 deletion5′-T ATT TGT AAA ACT GCG AGA TTA CTG TTA GTG TTG TAT GGT GCG GCC GCA TAG GCC ACT AG-3′ Open table in a new tab Gap1 activity was determined by measuring incorporation of 20 μm14C-labeled citrulline as described by Grenson (29Grenson M. Biochim. Biophys. Acta. 1966; 127: 339-346Crossref PubMed Scopus (97) Google Scholar). To avoid competitive inhibition of citrulline transport by glutamine, cells grown on glutamine medium were filtered, washed, and transferred to preheated proline medium just before the transport assay. The permease was inactivated by adding preheated (NH4)2SO4 to the culture (final concentration, 10 mm). Crude cell extracts (9Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar) and membrane-enriched preparations were prepared as previously described (10Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). In Western blot experiments, protein concentrations were estimated by densitometry of the Pma1 signal (ImageMaster1D,Amersham Pharmacia Biotech). Equal quantities of protein were loaded on an 8% SDS-polyacrylamide gel in a Tricine system (30Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). After transfer to a nitrocellulose membrane (Schleicher and Schüll), the proteins were probed with polyclonal antibodies raised against Gap1 (1: 10 000) or Pma1 (1: 1 000). Primary antibodies were detected with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Amersham Pharmacia Biotech) followed by enhanced chemiluminescence (Roche Molecular Biochemicals). The bul1Δ, bul2Δ, and npr1Δ null mutations were constructed by the polymerase chain reaction-based gene deletion method (31Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). Plasmid pUG6 (32Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1372) Google Scholar) served as template to generate DNA fragment loxP npr1::KanMX4 loxP with primers DEL1 and DEL2, DNA fragment loxP bul1::KanMX4 loxPwith primers D5-bul1 and D3-bul1 (Table II), and DNA fragment loxP bul2::KanMX4 loxP with primers D5-bul2 and D3-bul2 (Table II). Excision of the KanMX4 cassette was performed by transformation of yeast cells with plasmid pSH47 carrying the Cre recombinase gene (32Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1372) Google Scholar) and inducing Creexpression for 2 h. Cells were transformed by the lithium method (33Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) as modified by Gietz et al. (34Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). In wild-type cells grown on a medium containing urea or proline as sole nitrogen source, newly synthesized Gap1 is sorted from the Golgi to the plasma membrane where it accumulates in an active and stable form. In contrast, in mutant cells lacking the Npr1 kinase, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This direct sorting of Gap1 to the vacuole also occurs when wild-type cells are grown on a medium containing either glutamate (20Roberg K.J. Rowley N. Kaiser C.A. J. Cell Biol. 1997; 137: 1469-1482Crossref PubMed Scopus (161) Google Scholar) or NH4+ (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) as sole nitrogen source, this being consistent with Npr1 being inactive under these favorable nitrogen supply conditions (35Schmidt A. Beck T. Koller A. Kunz J. Hall M.N. EMBO J. 1998; 17: 6924-6931Crossref PubMed Scopus (263) Google Scholar). It was previously reported that loss of Gap1 activity in npr1 mutants is suppressed by the npi1 mutation (17Grenson M. Eur. J. Biochem. 1983; 133: 141-144Crossref PubMed Scopus (77) Google Scholar, 18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This mutation results in a markedly reduced level of the HECT-type ubiquitin ligase Npi1/Rsp5, leading to non-ubiquitination of Gap1 (9Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar, 10Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, 36Springael J.Y. De Craene J.O. Andre B. Biochem. Biophys. Res. Commun. 1999; 257: 561-566Crossref PubMed Scopus (47) Google Scholar). These observations raise the interesting possibility that direct sorting of Gap1 from the late secretory pathway to the vacuole involves ubiquitination of the permease. To investigate this possibility, we monitored the fate of newly synthesized Gap1 in wild-type, npr1Δ,npi1, and npr1Δ npi1 cells (Fig.1 A). The cells were first grown on glutamine medium to repress transcription of the GAP1 gene, then transferred to proline medium to relieve repression. In the wild type, this resulted in the appearance of a high intensity Gap1 signal on immunoblots and of high Gap1 activity in citrulline uptake assays (Fig. 1 A). The npi1strain displayed a similar phenotype. In the npr1Δ mutant, in keeping with the observation that Gap1 is directly sorted from the secretory pathway to the vacuole (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), no Gap1 activity was measured and only a low quantity of Gap1 was detectable after the cells were shifted to proline medium. The npr1Δ npi1 strain displayed a phenotype similar to that observed with the wild-type and npi1 strains, indicating that in the double mutant, neosynthesized Gap1 is targeted to the cell surface rather than to the vacuole. To confirm this result, we placed the GAP1 gene under the control of the galactose-inducible GAL1 promoter and monitored the neosynthesis of Gap1 by adding galactose to cells grown on a raffinose-proline medium (Fig. 1 B). As expected, this resulted in the progressive increase of Gap1 activity in the wild type, indicating that Gap1 was delivered to the plasma membrane, whereas the permease remained inactive in the npr1Δmutant. In the npr1Δ pep12Δ double mutant lacking the t-SNARE (target-soluble N-ethylmaleimide attachment protein receptor) protein Pep12 (required for transport of proteins from the Golgi to the late endosome/pre-vacuolar compartment) (37Becherer K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. Cell. 1996; 7: 579-594Crossref PubMed Scopus (253) Google Scholar), Gap1 activity gradually appeared after galactose addition in a manner similar to that in the pep12Δ mutant. This confirms that the pep12Δ mutation largely suppresses the effect of the npr1Δ mutation, even though the activity in the pep12Δ strain is lower than in the wild type (18De Craene J.-O. Soetens O. André B. J. Biol. Chem. 2001; 276: 43939-43948Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The same phenotype was observed in the npr1Δ npi1 strain (Fig.1 B). These results confirm those of Fig. 1 A and show that sorting of Gap1 from the late secretory pathway to the vacuole requires the ubiquitin ligase Npi1/Rsp5. They also show that when Npi1/Rsp5 is lacking, at least part of the neosynthesized Gap1 is rerouted to the plasma membrane. To further assess the role of ubiquitin in the direct sorting of Gap1 from the Golgi to the vacuole, we monitored the fate of neosynthesized Gap1 in npr1Δ cells also lacking the Npi2/Doa4 ubiquitin hydrolase. This enzyme facilitates ubiquitin recycling from proteasome-targeted substrates (38Swaminathan S. Amerik A.Y. Hochstrasser M. Mol. Biol. Cell. 1999; 10: 2583-2594Crossref PubMed Scopus (240) Google Scholar). In mutants affected in the Npi2/Doa4 ubiquitin hydrolase, the internal pool of ubiquitin is reduced severalfold (8Galan J. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (323) Google Scholar, 19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar, 39Terrell J. Shih S. Dunn R. Hicke L. Mol. Cell. 1998; 1: 193-202Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar); this impairs ubiquitination and the subsequent down-regulation of Gap1, which normally occur when NH4+ is added to proline-grown cells (19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar). Wild-type, npi2,npr1Δ, and npr1Δ npi2 strains were grown on glutamine medium and then shifted to proline medium (Fig.2). As expected, Gap1 remained inactive in the npr1Δ strain, and the quantity of Gap1 detected was much lower than in the wild type. In the npr1Δ npi2strain, Gap1 was as active as in the wild type, and an even higher amount of Gap1 was detected after the cells were shifted to proline medium, indicating that the npi2 mutation results in rerouting of neosynthesized Gap1 to the plasma membrane. In the npr1Δ npi2 strain overexpressing ubiquitin, a phenotype similar to that of the npr1Δ strain was observed, confirming that the effect of the npi2 mutation can be overcome by increasing the internal ubiquitin pool. Hence, direct sorting to the vacuole of neosynthesized Gap1 in npr1Δcells requires a normal pool of ubiquitin, and if this pool is too limiting, Gap1 is rerouted to the plasma membrane. The data presented above show that direct sorting to the vacuole of newly synthesized Gap1, as occurs in the npr1Δ mutant, requires normal levels of both Npi1/Rsp5 ubiquitin ligase and monomeric ubiquitin. These results suggest that ubiquitination of Gap1 could be required for its sorting to the vacuole. To test this hypothesis, experiments were conducted to isolate a mutant form of Gap1 resistant to ubiquitination. Previous work has identified the lysine residues of several permeases to which ubiquitin is attached (11Beck T. Schmidt A. Hall M.N. J. Cell Biol. 1999; 146: 1227-1238Crossref PubMed Scopus (251) Google Scholar, 13Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar, 14Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar). In the case of the uracil permease, for instance, ubiquitin is covalently linked to two lysine residues in a PEST region at the extreme N terminus (positions 38 and 41) (40Marchal C. Haguenauer-Tsapis R. Urban-Grimal D. Mol. Cell. Biol. 1998; 18: 314-321Crossref PubMed Scopus (108) Google Scholar). When these residues are mutated, ubiquitination and endocytosis of the permease are impaired. Each residue, furthermore, is subject to poly-ubiquitination, the ubiquitin moieties of the poly-ubiquitin chains being linked via the Lys63 residue of ubiquitin (41Marchal C. Haguenauer-Tsapis R. Urban-Grimal D. J. Biol. Chem. 2000; 275: 23608-23614Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In the case of the tryptophan permease Tat2, a protein homologous in sequence to Gap1, mutation of the five lysine residues present in the 31 N-terminal amino acids preceding the first transmembrane domain were needed to protect the permease against down-regulation induced by rapamycin treatment (11Beck T. Schmidt A. Hall M.N. J. Cell Biol. 1999; 146: 1227-1238Crossref PubMed Scopus (251) Google Scholar). These observations prompted us to mutagenize lysine residues present in the cytosolic N terminus of Gap1. Two Gap1 mutants were thus constructed in which lysine residues were replaced with arginine, respectively, at positions 9 and 16 (Gap1K9K16) and positions 51, 56, 60, and 63 (Gap1K51–K63) (Fig.3 A). The gap1Δstrain" @default.
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• W2080442977 title "Ubiquitin Is Required for Sorting to the Vacuole of the Yeast General Amino Acid Permease, Gap1" @default.
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