Ubiquitin Is Required for Sorting to the Vacuole of the Yeast General Amino Acid Permease, Gap1

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. A. J. Cell Biol. PubMed Scopus Google Scholar), the permease J. J. 1997; PubMed Google Scholar), and the J. 2000; PubMed Scopus Google Scholar). of of proteins shown to the ubiquitin-conjugating enzymes by the and an ubiquitin by the essential Haguenauer-Tsapis R. J. Biol. 2000; PubMed Google Scholar). Ubiquitin shown to an endocytosis signal in the of two two residues and L. EMBO J. 2000; PubMed Scopus Google Scholar). the protein of the endocytosis involved in ubiquitin recognition also remains as to ubiquitin also plays a role in the late of endocytosis and plasma membrane proteins of to the vacuole. we have the role of ubiquitin in the of the general amino acid permease which is by proline or of nitrogen the is to M. J. PubMed Scopus Google Scholar), and the synthesized Gap1 permease accumulates at the plasma membrane in an and stable M. J. PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of NH4+ preferential source of Gap1 is by endocytosis and to the vacuole for degradation. of Gap1 is essential to this 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 mutant, which an of the ubiquitin Npi1/Rsp5, or the mutant lacking the Gap1 is not ubiquitinated and at the plasma membrane after NH4+ J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google J.Y. Haguenauer-Tsapis R. Andre B. J. Cell 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 the ubiquitin to the lysine 63 of the preceding ubiquitin J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar) the lysine poly-ubiquitin Gap1 is required for down-regulation of the permease at a J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). The of newly synthesized Gap1 in the late secretory pathway is also nitrogen proline or neosynthesized Gap1 is to the plasma but in a J. Cell Biol. 1997; PubMed Scopus Google Scholar) or NH4+ B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as the nitrogen Gap1 is sorted from the Golgi to the vacuole without passing the A on proline with cells lacking Npr1, a protein kinase both cell-surface and Gap1 B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and by when nitrogen are A. M. T. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we show that Gap1 is ubiquitinated on two lysine residues in its N at 9 and the Gap1K9K16 in which both lysine residues are mutated, we show that ubiquitination of Gap1 is required not only for down-regulation of the protein at the but also for direct sorting of the protein from the late secretory pathway to the vacuole, as occurs in an npr1Δ mutant. We show that ubiquitination and degradation of both cell-surface and Gap1 Bul1 and Bul2, two proteins interacting with the ubiquitin this by J. Cell Biol. PubMed Scopus Google Scholar) that sorting of Gap1 to the vacuole its and that the role of Bul1 and is to this in this are with J. M. J. PubMed Scopus Google Scholar). in M. J. Mol. Biol. PubMed Scopus Google Scholar) with as the source when In proline the nitrogen In of Gap1 cells on or NH4+ and to proline to In ubiquitin cells on and to nitrogen without NH4+ or amino and to of ubiquitin. The a yeast the control of the M. A. A. PubMed Scopus Google Scholar). The the ubiquitin in which lysine 63 or lysine residues 29, 48, and 63 are replaced by arginine are from T. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). C. Andre B. Mol. Microbiol. 1997; PubMed Scopus Google Scholar) is on the M. L. PubMed Scopus Google Scholar). to M. L. PubMed Scopus Google in this B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M. J. PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google in a of the on as by the The for each are in by in this in a Gap1 by of as by M. PubMed Scopus Google Scholar). of by cells on and to proline the The permease by to the (9Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar) and as previously J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). In protein by of the signal of protein on an in a H. PubMed Scopus Google Scholar). to a membrane and the proteins with Gap1 or with followed by The and npr1Δ by the chain A. A. R. 1994; PubMed Scopus Google Scholar). T. J. 1996; PubMed Scopus Google Scholar) as to with and and and with and of the by of yeast cells with the T. J. 1996; PubMed Scopus Google Scholar) and for by the H. K. A. J. PubMed Google Scholar) as by A. PubMed Scopus Google Scholar). In cells on a or proline as nitrogen newly synthesized Gap1 is sorted from the Golgi to the plasma membrane accumulates in an and stable In in mutant cells lacking the neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing the plasma membrane B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This direct sorting of Gap1 to the vacuole also occurs when cells are on a J. Cell Biol. 1997; PubMed Scopus Google Scholar) or NH4+ B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as nitrogen this with nitrogen A. T. A. J. EMBO J. 1998; PubMed Scopus Google Scholar). previously that of Gap1 in is by the M. J. PubMed Scopus Google Scholar, B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This results in a of the ubiquitin Npi1/Rsp5, to 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, J.Y. De Andre B. PubMed Scopus Google Scholar). These the that direct sorting of Gap1 from the late secretory pathway to the vacuole ubiquitination of the this we the of newly synthesized Gap1 in and npr1Δ cells The cells on to of the to proline to In the this in the of a Gap1 signal on and of Gap1 in The a In the npr1Δ mutant, in with the that Gap1 is sorted from the secretory pathway to the vacuole B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), Gap1 and only a of Gap1 after the cells to proline The npr1Δ a to that with the and that in the mutant, neosynthesized Gap1 is to the to the vacuole. this we the the control of the and the of Gap1 by to cells on a this in the of Gap1 in the that Gap1 to the plasma the permease in the In the npr1Δ mutant lacking the protein protein for of proteins from the Golgi to the late Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar), Gap1 after in a to that in the mutant. This that the the of the npr1Δ the in the is in the B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the npr1Δ These results of A and show that sorting of Gap1 from the late secretory pathway to the vacuole the ubiquitin also show that when is at of the neosynthesized Gap1 is rerouted to the plasma membrane. the role of ubiquitin in the direct sorting of Gap1 from the Golgi to the vacuole, we the of neosynthesized Gap1 in npr1Δ cells also lacking the ubiquitin This ubiquitin from M. Mol. Biol. Cell. PubMed Scopus Google Scholar). In in the ubiquitin the of ubiquitin is (8Galan J. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (323) Google Scholar, J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar, J. R. L. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google this ubiquitination and the down-regulation of Gap1, which occur when NH4+ is added to proline-grown cells J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). and npr1Δ on and to proline Gap1 in the npr1Δ and the of Gap1 in the In the npr1Δ Gap1 as as in the and an of Gap1 after the cells to proline that the results in of neosynthesized Gap1 to the plasma membrane. In the npr1Δ a to that of the npr1Δ that the of the by the ubiquitin direct sorting to the vacuole of neosynthesized Gap1 in a of and this is Gap1 is rerouted to the plasma membrane. The show that direct sorting to the vacuole of newly synthesized Gap1, as occurs in the npr1Δ mutant, of both ubiquitin and ubiquitin. These results that ubiquitination of Gap1 required for its sorting to the vacuole. this to a mutant of Gap1 to the lysine residues of several to which ubiquitin is (11Beck T. A. J. Cell Biol. PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar, Haguenauer-Tsapis R. J. Biol. 2000; PubMed Google Scholar). In the of the uracil for ubiquitin is linked to two lysine residues in a at the N terminus (positions and C. Haguenauer-Tsapis R. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). residues are mutated, ubiquitination and endocytosis of the permease are is subject to the ubiquitin of the poly-ubiquitin linked the of ubiquitin C. Haguenauer-Tsapis R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the tryptophan permease a protein homologous in to Gap1, of the lysine residues in the amino preceding the to the permease down-regulation by (11Beck T. A. J. Cell Biol. PubMed Scopus Google Scholar). These to lysine residues in the cytosolic N terminus of Gap1 in which lysine residues replaced with at 9 and (Gap1K9K16) and and 63 The with the or the on cells on proline that both the and the fully Gap1 The of NH4+ to cells in the endocytosis of Gap1, as shown by of Gap1 followed by degradation of the as shown by of the Gap1 signal on J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). A with cells the that lysine residues at and 63 are not important for In the permease by the and stable after NH4+ and that the lysine at 9 and/or to down-regulation of the of each lysine we and In the the lysine at 9 or is replaced with showed that both of Gap1 are fully on proline NH4+ both that the lysine residues at 9 and are each to down-regulation of the Gap1K9 and Gap1K16 down-regulated more the Gap1 protein, that both lysine residues are required to We the ubiquitination of Gap1, and of we with J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). We cells cells the or with a the ubiquitin the control of the T. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). The signal in proline-grown cells Gap1 of a at at to of the permease J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). In with J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar), the of of Gap1 in the after NH4+ addition. also in cells the or that Gap1 mutant are in cells the the These show that Gap1 is ubiquitinated on the lysine residues at 9 and Gap1 is ubiquitinated on only two previously from at the of ubiquitin chain linked to Gap1 J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). in an of a ubiquitin mutant to of poly-ubiquitin Gap1 to or two ubiquitin J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). A with cells a ubiquitin in of that poly-ubiquitin chain on Gap1 through the lysine 63 of ubiquitin J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). The in show that of of ubiquitin in cells or a single accumulates the Gap1 In two the Gap1 signal in cells These results show that both lysine residues (positions 9 and are ubiquitinated with The in that of Gap1 at 9 and the permease at the plasma membrane to lysine residues of Gap1 are also required for direct sorting of neosynthesized permease from the late secretory pathway to the vacuole, we a with the or on a NH4+ at as to and to proline to Gap1 in the npr1Δ the The results the when the or in the Gap1 in cells the Gap1K9K16 the Gap1K9K16 in is rerouted to the plasma membrane in an This result and and that ubiquitination of Gap1 is essential to its direct sorting from the late secretory pathway to the vacuole. of a single lysine 9 or is for the sorting of Gap1 to the vacuole, the Gap1K9 and Gap1K16 both We poly-ubiquitin chain is required for the sorting of Gap1 to the vacuole. we the and of Gap1 neosynthesized in an npr1Δ ubiquitin or a ubiquitin mutant in which lysines at 29, 48, and 63 are replaced by arginine This ubiquitin is to poly-ubiquitin in T. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). of ubiquitin the of the in the npr1Δ Gap1 and not to after the of the cells from to Although a and of Gap1 when of ubiquitin in the npr1Δ the of the also by This that poly-ubiquitin chain is not essential to the direct sorting of Gap1 to the vacuole. Bul1 and are proteins shown in and to with the ubiquitin H. T. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, H. A. 1998; PubMed Scopus Google Scholar). is evidence that this a in the proteins and or all of the of the in Bul1 is not a of Npi1/Rsp5, that the proteins with in protein ubiquitination H. T. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, H. A. 1998; PubMed Scopus Google Scholar). the role of Bul1 and in Gap1 trafficking, and single and a mutant We the of the and on Gap1 and after the of cells on proline In the Gap1 the and degradation in the A and We the of Gap1 ubiquitination in this mutant. In with J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar), of of cells after NH4+ showed an of the to of Gap1 These in the mutant, that ubiquitination of Gap1 is in this mutant with the and single that Gap1 is ubiquitinated and to down-regulation in the Bul1 and proteins to a essential to Gap1 ubiquitination and down-regulation of cell-surface We the direct sorting of neosynthesized Gap1 to the vacuole in an npr1Δ also the proteins. The npr1Δ and an npr1Δ mutant on and to proline Gap1 in the In the Gap1 of the npr1Δ as as that of the that neosynthesized Gap1 is rerouted to the plasma membrane in this mutant. This result that a in Gap1 ubiquitination not only Gap1 at the plasma membrane down-regulation but also Gap1 neosynthesized in an npr1Δ from sorted to the vacuole. In this we show that ubiquitination of the yeast Gap1 permease on two lysine residues in the N terminus of the protein (positions 9 and a shown to cytosolic J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, each lysine a chain of at two ubiquitin linked to each the lysine 63 of ubiquitin. A for the uracil permease which is by on residues and The Gap1K9K16 in which both lysine residues are replaced with is fully endocytosis and which occur when NH4+ is added to cells growing on proline or as the nitrogen The Gap1K9 and Gap1K16 in which a single lysine is are down-regulated after This that ubiquitination of Gap1 on a single lysine 9 or is for endocytosis and degradation. down-regulation of both Gap1K9 and Gap1K16 is Gap1, that ubiquitination of Gap1 on both lysines is required for We have also shown in to the ubiquitin at of the two proteins to with Npi1/Rsp5, Bul1 or H. T. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, H. A. 1998; PubMed Scopus Google Scholar), is essential to ubiquitination and down-regulation of results that a and at of the proteins of ubiquitin to lysine residues 9 and of to of Gap1 the of ubiquitin J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar) is also by the or ubiquitin is Our show that ubiquitin plays an essential role in pathway of Gap1 trafficking, direct sorting of neosynthesized Gap1 from the late secretory pathway to the vacuole. This direct sorting to the vacuole occurs in cells in which is in the npr1Δ mutant on proline and in cells growing nitrogen B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 1997; PubMed Scopus Google Scholar). We have shown here that ubiquitination of Gap1 is a result of an or Gap1 the permease is rerouted to the plasma membrane. We that in the on proline Gap1 is to the plasma but that of the results in sorting of Gap1 to the vacuole B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This sorting ubiquitination of the permease on at of the two lysine residues 9 and In the of Gap1 ubiquitination in an npr1Δ mutant, Gap1 is to the plasma membrane. The by which the kinase the sorting of Gap1 to the of Gap1 is not on Npr1, that the of is B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The role of ubiquitin in the of Gap1 to the vacuole also remains Ubiquitin as a signal for Gap1 for the vacuole, for from the late Golgi and/or by of the membrane to a M. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). this ubiquitination is Gap1 from this to the plasma membrane. Our to the growing of evidence that ubiquitin plays an important role in the sorting of membrane proteins in the and/or late Golgi Cell Biol. 2000; PubMed Scopus Google Scholar). In the an ubiquitin and the by the of the ubiquitin with the late J. M. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). Furthermore, the for the to for of the vacuolar pathway J. M. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). In study, a mutant of the tryptophan permease of ubiquitin to that to direct sorting of the permease from the late secretory pathway to the vacuole (11Beck T. A. J. Cell Biol. PubMed Scopus Google Scholar). is for of that Tat2 and Gap1 are by A. T. A. J. EMBO J. 1998; PubMed Scopus Google Scholar). with a role for ubiquitin in sorting to the vacuole is the that the protein by the T. C. Mol. Cell. Biol. PubMed Scopus Google Scholar) and its by the M. 2000; PubMed Scopus Google Scholar) both a to that in the ubiquitin-conjugating enzymes This the to which ubiquitin is linked in enzymes Genet. 1997; 16: PubMed Scopus Google Scholar, C.P. J. Mol. 1997; PubMed Scopus Google Scholar). in the for their to to the a mutant Ste2 which at a is from the secretory pathway to the vacuole for degradation T. C. Mol. Cell. Biol. PubMed Scopus Google Scholar). In mutant the which the and is to the M. 2000; PubMed Scopus Google Scholar). These are of to neosynthesized Gap1, rerouted to the plasma membrane when its sorting to the vacuole is by In the of the epidermal growth factor receptor ubiquitin is to a role in sorting of to the its to the H. L. B. 1998; PubMed Scopus Google Scholar). In the mutant, is to the in the M. 2000; PubMed Scopus Google Scholar). Finally, that the ubiquitin of yeast sorting of the Ste6 ABC from membrane to the vacuole A. R. Mol. Biol. Cell. PubMed Scopus Google Scholar). Our results also show that the and involved in Gap1 ubiquitination control the of both cell-surface and the ubiquitin the Bul1 and and the lysine residues at 9 and in Gap1 are required for both down-regulation of Gap1 at the and sorting to the vacuole of Gap1 in the late secretory also both of Gap1 by their sorting to the vacuolar degradation pathway nitrogen B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, in the of Gap1, a Gap1 in an down-regulation of cell-surface Gap1 but of ubiquitin to Gap1 J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google C. Andre B. Mol. Microbiol. 1997; PubMed Scopus Google Scholar). We several to for the mechanism the and in both cytosolic of Gap1 at both the plasma membrane and membrane to ubiquitin on lysines of Gap1, a the sorting of the permease to the vacuolar degradation the of Gap1 endocytosis not an at the of the and secretory Gap1 ubiquitinated or In the sorted to the vacuole for degradation when is nitrogen In the to the occur when is nitrogen The the of neosynthesized Gap1 this ubiquitination to sorting to the vacuole nitrogen and to to the plasma membrane nitrogen Finally, the of ubiquitination by Gap1 also the of the we previously that when cells on nitrogen proline or a of the Gap1 is but the of NH4+ or of by poly-ubiquitin J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). of Gap1 at the or late secretory pathway of the permease to an sorting Gap1 to its sorting to the vacuole the pathway nitrogen or to its to the plasma membrane nitrogen Our at the of this that neosynthesized Gap1 is to the vacuole in cells and that this a J. Cell Biol. PubMed Scopus Google Scholar). These that Gap1 is in cells ubiquitin. In the of Gap1 to and of Gap1 These from of Gap1 only the of NH4+ to or cells J.Y. Haguenauer-Tsapis R. Andre B. J. Cell PubMed Google Scholar). Furthermore, we not of Gap1 in the we show an essential role of poly-ubiquitin in sorting of Gap1 to the vacuole This in cells in the poly-ubiquitin is required for sorting of Gap1 to the vacuole only cells a Finally, the J. Cell Biol. PubMed Scopus Google Scholar) that Bul1 and as enzymes of These that of of Gap1 down-regulation of Gap1, show that a is essential to endocytosis followed by vacuolar degradation of Gap1 A and are to the role of poly-ubiquitin in control of Gap1 These studies a of the role of ubiquitin in the of membrane proteins. We and for and and for their