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• W2014533300 abstract "In Saccharomyces, the addition of glucose induces a rapid degradation of maltose permease that is dependent on endocytosis and vacuolar proteolysis (Medintz, I., Jiang, H., Han, E. K., Cui, W., and Michels, C. A. (1996) J. Bacteriol. 178, 2245–2254). Here we report on the role of ubiquitin conjugation in this process. Deletion of DOA4, which causes decreased levels of available ubiquitin, severely decreases the rate of glucose-induced proteolysis, and this is suppressed by the overproduction of ubiquitin. Overexpression of ubiquitin in an endocytosis-deficient end3-ts strain results in the glucose-stimulated accumulation of a larger molecular weight species of maltose permease, which we demonstrate is a ubiquitin-modified form of the protein by utilizing two ubiquitin alleles with different molecular weights. The size of this ubiquitinated species of maltose permease is consistent with monoubiquitination. A promoter mutation that reduces expression ofRSP5/NPI1, a postulated ubiquitin-protein ligase, dramatically reduces the rate of glucose-induced proteolysis of maltose permease. The role of various ubiquitin-conjugating enzymes was investigated using strains carrying mutant alleles ubc1Δubc4Δ, ubc4Δ ubc5Δ,cdc34-ts2/ubc3, and ubc9-ts. Loss of these functions was not shown to effect glucose-induced proteolysis of maltose permease, but loss of Ubc1, -4, and -5 was found to inhibit maltose permease expression at the post-transcriptional level. In Saccharomyces, the addition of glucose induces a rapid degradation of maltose permease that is dependent on endocytosis and vacuolar proteolysis (Medintz, I., Jiang, H., Han, E. K., Cui, W., and Michels, C. A. (1996) J. Bacteriol. 178, 2245–2254). Here we report on the role of ubiquitin conjugation in this process. Deletion of DOA4, which causes decreased levels of available ubiquitin, severely decreases the rate of glucose-induced proteolysis, and this is suppressed by the overproduction of ubiquitin. Overexpression of ubiquitin in an endocytosis-deficient end3-ts strain results in the glucose-stimulated accumulation of a larger molecular weight species of maltose permease, which we demonstrate is a ubiquitin-modified form of the protein by utilizing two ubiquitin alleles with different molecular weights. The size of this ubiquitinated species of maltose permease is consistent with monoubiquitination. A promoter mutation that reduces expression ofRSP5/NPI1, a postulated ubiquitin-protein ligase, dramatically reduces the rate of glucose-induced proteolysis of maltose permease. The role of various ubiquitin-conjugating enzymes was investigated using strains carrying mutant alleles ubc1Δubc4Δ, ubc4Δ ubc5Δ,cdc34-ts2/ubc3, and ubc9-ts. Loss of these functions was not shown to effect glucose-induced proteolysis of maltose permease, but loss of Ubc1, -4, and -5 was found to inhibit maltose permease expression at the post-transcriptional level. ubiquitin carrier protein hemagglutinin. Glucose regulates maltose transport in Saccharomyces at several levels. It blocks transcription of the maltose permease gene by multiple mechanisms cumulatively referred to as glucose repression (2Hu Z. Nehlin J.O. Ronne H. Michels C.A. Curr. Genet. 1995; 28: 258-266Crossref PubMed Scopus (84) Google Scholar), and it inactivates maltose permease by a process referred to as glucose-induced inactivation or catabolite inactivation (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 3Lucero P. Lagunas L. FEMS Microbiol. Lett. 1997; 147: 273-277Crossref PubMed Google Scholar). Together, these processes allow for the rapid shift from maltose to glucose fermentation. Previously, we showed that glucose-induced inactivation of maltose permease consists of two apparently independent processes: the proteolysis of maltose permease protein and the rapid inhibition of maltose transport activity, which occurs even faster than can be explained by loss of the protein alone (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). Molecular genetic analysis using mutations in END3, REN1/VPS2,PEP4, and PRE1 and PRE2 demonstrated that the proteolysis of maltose permease is dependent on endocytosis, vesicle sorting, and vacuolar proteolysis and is independent of the proteasome. Studies of a variety of different nutrient transporters suggest that the inactivation and/or degradation of permeases is a generalized mechanism used to respond to changes in nutrient availability from less desirable nutrient sources or starvation conditions to preferred nutrients and rich medium. The general amino acid permease Gap1 protein is inactivated by the addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source (5Stanbrough M. Magasanik B. J. Bacteriol. 1995; 177: 94-102Crossref PubMed Google Scholar, 6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar). Inactivation occurs as a 2-fold process with enzymatic inactivation by phosphorylation preceding degradation of the permease (5Stanbrough M. Magasanik B. J. Bacteriol. 1995; 177: 94-102Crossref PubMed Google Scholar, 6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar). The high affinity Pho84 phosphate transporter undergoes rapid degradation once the supply of phosphate and/or carbon source is exhausted (7Martinez P. Zvyagilakaya R. Allard P. Persson B.L. J. Bacteriol. 1998; 180: 2253-2256Crossref PubMed Google Scholar). Uracil permease (Fur4p) is phosphorylated on serine residues at the plasma membrane and is rapidly degraded under adverse growth conditions (8Galan J.M. Moreau V. Andre B. Volland C. Haguenauer-Tsapis R. J. Biol. Chem. 1996; 271: 10946-10952Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). A common feature of the degradation of the maltose, galactose, uracil, and general amino acid permeases is that all are mediated by endocytosis and subsequent transport to the vacuole, the site of degradation. Ubiquitination has been implicated as the mechanism marking these proteins and several others for rapid endocytosis and selective degradation (4Horak J. Wolf D.H. J. Bacteriol. 1997; 179: 1541-1549Crossref PubMed Google Scholar, 6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar,8Galan J.M. Moreau V. Andre B. Volland C. Haguenauer-Tsapis R. J. Biol. Chem. 1996; 271: 10946-10952Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 9Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar, 10Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 11Kolling R. Hollenberg C.P. EMBO J. 1994; 13: 3261-3271Crossref PubMed Scopus (271) Google Scholar). We report here that ubiquitination of the maltose permease occurs in response to glucose and explore the cellular components involved in this process. Ubiquitination of Saccharomyces Ste2 protein, α-factor receptor, is required for its ligand-stimulated endocytosis and vacuolar proteolysis (10Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). END4 mutations inhibit endocytosis of α-factor and stimulate the appearance of multiubiquitinated species. A sequence in the C-terminal cytoplasmic domain of Ste2p, SINNDAKSS (12Rohrer H. Benedetti H. Zanolari B. Riezman H. Mol. Biol. Cell. 1993; 4: 511-521Crossref PubMed Scopus (101) Google Scholar), is sufficient to stimulate endocytosis, but mutation of the Lys in this target sequence to Arg inhibits ligand-stimulated ubiquitination and degradation. These results clearly implicate ubiquitination in receptor targeting to endocytosis. Ubiquitination also is required for endocytosis of yeast uracil permease (8Galan J.M. Moreau V. Andre B. Volland C. Haguenauer-Tsapis R. J. Biol. Chem. 1996; 271: 10946-10952Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) and probably the galactose transporter, Gal2p (4Horak J. Wolf D.H. J. Bacteriol. 1997; 179: 1541-1549Crossref PubMed Google Scholar). Additional studies also have implicated ubiquitination as a signal for the endocytosis and vacuolar degradation of other plasma membrane proteins including mammalian peptide hormone receptors (reviewed in Ref. 13Hicke L. FASEB J. 1997; 11: 1215-1226Crossref PubMed Scopus (229) Google Scholar). The yeast ABC transporter Ste6 accumulates in a ubiquitinated form in the plasma membrane of strains that are deficient in endocytosis (11Kolling R. Hollenberg C.P. EMBO J. 1994; 13: 3261-3271Crossref PubMed Scopus (271) Google Scholar). In strains that have normal endocytotic functions, this protein is generally found associated with internal membranes. Another protein from this same family of yeast transporters, the multidrug transporter Pdr5, also is ubiquitinated prior to endocytosis and degradation in the vacuole, suggesting that ubiquitination may trigger the endocytosis of this short lived protein (14Egner R. Kuchler K. FEBS Lett. 1996; 378: 177-181Crossref PubMed Scopus (102) Google Scholar). Similar results have been reported for the human fibroblast growth factor receptor (15Strous G.J. van Kerkhof P. Govers R. Ciechanover A. Schwartz A.L. EMBO J. 1996; 15: 3806-3812Crossref PubMed Scopus (265) Google Scholar). Moreover, many other plasma membrane receptor proteins are found as ubiquitin conjugates including the lymphocyte homing receptor, the platelet-derived growth factor receptor, the c-Kit receptor, and the mammalian immunoglobulin E receptor (16Siegelman M. Bond M.W. Gallatin W.M. St. John T. Smith H.T. Fried V.A. Weissman I.L. Science. 1986; 231: 823-829Crossref PubMed Scopus (205) Google Scholar, 17Mori S. Heldin C.H. Claesson-Welsh L. J. Biol. Chem. 1992; 267: 6429-6434Abstract Full Text PDF PubMed Google Scholar, 18Miyazawa K. Toyama K. Gotoh A. Hendrie P.C. Mantel C. Broxmeyer H.E. Blood. 1994; 83: 137-145Crossref PubMed Google Scholar, 19Paolini R. Kinet J.P. EMBO J. 1993; 12: 779-786Crossref PubMed Scopus (108) Google Scholar). In this study, we used molecular genetic analysis to explore the role of ubiquitin in the glucose-induced inactivation of the maltose permease. Our results indicate that loss of free ubiquitin, via aDOA4 null mutation, impairs the glucose-induced proteolysis of maltose permease and that the effects of the doa4Δ null mutation can be suppressed by the overexpression of ubiquitin. We demonstrate that the maltose permease exists as a ubiquitinated species and that the amount of this ubiquitinated species increases dramatically upon the addition of glucose to maltose fermenting cells. Rsp5/Npi1 ubiquitin-protein ligase is implicated in the proteolysis of maltose permease. Mutations in UBC1, UBC4, andUBC5 encoding ubiquitin conjugation enzymes UBC (ubiquitin carrier proteins; E21enzymes), in combination were found to dramatically decrease the level of maltose permease expressed, apparently by affecting a post-transcriptional process but not glucose-induced proteolysis. The Saccharomyces cerevisiae strains used in this study and their relevant genotypes are listed in Table I. Plasmid pDOA4–8 carries the wild-type allele of DOA4. Plasmid YEp96 (pCUP1-Ub) contains UBI4 encoding ubiquitin expressed from the copper-inducible CUP1 promoter, and YEp105 (pCUP1-mycUb) contains a c-myc-tagged ubiquitin allele also expressed from the CUP1 promoter (20Ecker D.J. Khan M.I. Marsh J. Butt T.R. Crooke S.T. J. Biol. Chem. 1987; 262: 3524-3527Abstract Full Text PDF PubMed Google Scholar, 21Hochstrasser M. Ellison M.J. Chau V. Varshvsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4604-4610Crossref Scopus (197) Google Scholar). These plasmids were obtained from Mark Hochstrasser (University of Chicago). Plasmid pUN70 serves as a yCP vector control (22Elledge S.J. Davis R.W. Gene (Amst.). 1988; 70: 303-312Crossref PubMed Scopus (163) Google Scholar) as does plasmid yATAG200 (pCUP1-vector), which contains a CUP1 promoter without any fused gene sequence.Table IS. cerevisiae strains used in this studyStrainGenotypeSourceCMY1001MAT a MAL61/HA MAL12 MAL13 GAL leu2 ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 DOA4Ref. 1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google ScholarCMY1004end3-ts (isogenic to CMY1001)Ref. 1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google ScholarPMY270MATα doa4Δ1::LEU2 his3-Δ200 leu2–3,112 ura3–52 lys2–801 trp1–1 MAL31 MAL32P. McGrawCMY1025doa4Δ1::LEU2 his3-Δ200 ura3–52 lys2–801 trp1 MAL61/HA MAL12 MAL13 MAL31 MAL32This study23346cMAT a ura3 NPI1Ref. 6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar27038aMAT a ura3 npilRef. 6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google ScholarMGG15MAT a cdc34–2ts ura3–52 his3-Δ200Ref. 37Banerjee A. Deshaies R.J. Chau V. J. Biol. Chem. 1995; 270: 26209-26215Abstract Full Text Full Text PDF PubMed Scopus (37) Google ScholarMHY501MAT a his3-Δ200 leu2–3,112 lys2–801 trp1–1Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarMHY498ubc4-Δ1::HIS3 (isogenic to MHY 501)Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarMHY499ubc5-Δ1::LEU2 (isogenic to MHY 501)Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarMHY509ubc1-Δ1::HIS3(isogenic to MHY 501)Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarMHY508ubc4-Δ1::HIS3 ubc5-Δ1::LEU2(isogenic to MHY 501)Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarMHY519ubc1-Δ1::URA3 ubc4-Δ1::HIS3(isogenic to MHY 501)Ref. 34Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (354) Google ScholarFM394MAT a his3-Δ200 leu2–3,112 ura3–52 lys2–801 trp1–1(am)Ref. 45Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google ScholarFM395MATα his3-Δ200 leu2–3,112 ura3–52 lys2–801 trp1–1(am) ubc9Δ::TRP1 leu2::ubc9Pro-Ser::LEU2Ref. 45Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar Open table in a new tab Plasmids pRS416-MAL61/HA, pUN70-MAL61/HA, pRS415-MAL61/HA, and pUN30-MAL61/HA all carry the HA-tagged maltose permease under the control of its native promoter (22Elledge S.J. Davis R.W. Gene (Amst.). 1988; 70: 303-312Crossref PubMed Scopus (163) Google Scholar, 23Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Plasmids pUN90-MAL63, pUN30-MAL63, and YCP50-MAL63 all carry the MAL63 MAL- activator gene, required in many strains for maltose-induced expression of the MAL structural genes. Plasmid pADH1-MAL61 expressing the MAL61/HA gene from the constitutive ADH1 promoter was constructed as follows. Usingin vitro mutagenesis, an XhoI site was introduced into pUN30-MAL61/HA 12 base pairs upstream of the start codon of the permease gene MAL61/HA. The promoter sequence of this gene was removed by digestion withXhoI and SacI and replaced with the 400-base pairADH1 promoter, amplified from plasmid pGAD424 (CLONTECH Inc., Palo Alto, CA) by polymerase chain reaction. Strain CMY1025 is a maltose fermenting leucine+ haploid segregant from a diploid obtained by mating strains CMY1001 and PMY270, which carries adoa4Δ::LEU2 disruption (24Papa F.R. Hochstrasser M. Nature. 1993; 88: 4604-4610Google Scholar). Southern analysis using MAL61-specific probes revealed the presence two maltose permease genes, one at the MAL1 locus (MAL61/HA, derived from CMY1001) and a second (MAL31, derived from PMY270) mapping to the partially functional MAL3 locus encoding MAL31 (maltose permease) and MAL32 (maltase) (25Charron M.J. Dubin R.A. Michels C.A. Mol. Cell. Biol. 1986; 6: 3891-3899Crossref PubMed Scopus (73) Google Scholar). The standard inactivation assay protocol was used as described previously (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). Unless otherwise indicated, cells were grown at 30 °C to early log phase (A 6000.1–0.3) in YP (rich) or SM (selection) medium containing 2% maltose, harvested by filtration with cellulose filters, and resuspended in nitrogen starvation medium (1.74 g/liter of yeast nitrogen base without amino acids and ammonium sulfate) plus 2% (w/v) carbon source, usually glucose. At selected time intervals, cells were harvested by filtration for Western analysis and maltose transport assays. All values depicted in this study are the average of at least two experiments and were carried out in duplicate. Variation was less than 15%. Growth dilution was calculated as the A 600 at time 0 divided by the A 600 at time x. Maltose transport was measured by the uptake of 1 mm[14C]maltose as described previously (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 26Cheng Q. Michels C.A. Genetics. 1989; 123: 477-484Crossref PubMed Google Scholar). Transport assays were done in duplicate on at least duplicate cultures. Maltase activity was determined as described previously (27Dubin R.A. Needleman R.B. Gossett D. Michels C.A. J. Bacteriol. 1985; 164: 605-610Crossref PubMed Google Scholar). Maltase activity describes the nmol of p-nitrophenol α-d-glucopyranoside cleaved per mg of protein per min as measured spectrophotochemically. Cells were harvested, and total protein extracts were prepared by the methods described previously (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 28Davis N.G. Horecka J.L. Sprague Jr., G.F. J. Cell. Biol. 1993; 122: 53-65Crossref PubMed Scopus (192) Google Scholar). Equal amounts of total protein are loaded per well for comparison of time courses or relative protein levels. SDS-polyacrylamide gel electrophoresis analysis and detection were carried out for the HA-tagged Mal61 maltose permease (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). The intensity of the signal was quantitated by scanning films with a Beckman DU640 spectrophotometer, and relative Mal61/HA protein levels were determined by comparison of the area under the curve. Western blots were done in duplicate on all samples for duplicate experimental cultures, and densitometer quantitation of the relative protein levels was carried out twice for each sample lane (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). The yeast DOA4 gene encodes a ubiquitin hydrolase enzyme that functions late in the proteasomal degradation pathway by cleaving and recycling ubiquitin from substrate remnants still bound to protease (24Papa F.R. Hochstrasser M. Nature. 1993; 88: 4604-4610Google Scholar). Although Doa4p is only one of several species of ubiquitin hydrolase enzymes found inSaccharomyces, loss of the DOA4 gene product significantly decreases the rate of ubiquitin recycling and severely decreases levels of available ubiquitin. We used a doa4Δnull mutant strain to explore the dependence on ubiquitin of glucose-induced proteolyis of maltose permease. Glucose-induced inactivation of maltose permease was characterized in the doa4Δ null strain, CMY1025, and as a control in strain CMY1025 carrying the wild-type DOA4 gene on a CENplasmid. As is evident from Fig. 1(top two panels), the doa4mutant strain exhibits a dramatically decreased rate of glucose-induced proteolysis of Mal61/HA permease, to the extent that the loss of maltose permease protein parallels the growth of the culture (growth dilution). In comparison, in the DOA4 strain, maltose permease protein is degraded more rapidly than can be expected from growth alone. Table II indicates that the steady state rate of maltose transport in the doa4Δ strain is slightly higher (37%) than that of a strain expressing the wild-type DOA4 gene, which is consistent with the decrease in maltose permease turnover. Interestingly, despite the apparent lack of glucose-induced proteolysis of maltose permease in thedoa4Δ strain, glucose stimulates a decrease in maltose transport activity, indicating that the inhibition of transport activity occurs by a process that is independent of ubiquitin availability.Table IIMaltose transport rates of doa4 and rsp5/npi1 mutant strainsStrainRelevant genotypeTransport ratenmol/mg (dry wt)/minCMY1025 (pCUP1-vector)doa4Δ::LEU21.57aDetermined following a 4-h incubation in 0.1 mm copper sulfate.CMY1025 (pCUP1-Ub)doa4Δ::LEU20.80aDetermined following a 4-h incubation in 0.1 mm copper sulfate.CMY1025 (pUN70)doa4Δ::LEU21.72CMY1025 (pDOA4–8)doa4Δ::LEU21.2623346c (pRS416MAL61/HA)RSP5/NPI13.0327038a (pRS416MAL61/HA)rsp5/npil3.62All strains were grown in rich medium with 2% maltose to early log phase at 30 °C. Maltose transport rates were determined as described under “Materials and Methods.” See “Materials and Methods” for description of plasmids pCUP1-Ub (yEP96) and pCUP1-vector (yATAG200). All determinations are from two separate cultures, each assayed in duplicate. Variation is less than 15%.a Determined following a 4-h incubation in 0.1 mm copper sulfate. Open table in a new tab All strains were grown in rich medium with 2% maltose to early log phase at 30 °C. Maltose transport rates were determined as described under “Materials and Methods.” See “Materials and Methods” for description of plasmids pCUP1-Ub (yEP96) and pCUP1-vector (yATAG200). All determinations are from two separate cultures, each assayed in duplicate. Variation is less than 15%. In order to test the possibility that the ubiquitin deficiency in thedoa4Δ strain is responsible for the decreased rate of glucose-induced proteolysis of maltose permease, we determined whether overexpression of ubiquitin could overcome the loss of active ubiquitin recycling. Plasmid yEP96 (pCUP1-Ub), carrying the ubiquitin geneUBI4 fused to the copper-responsive promoter fromCUP1, was introduced into the doa4Δ mutant strain CMY1025 (20Ecker D.J. Khan M.I. Marsh J. Butt T.R. Crooke S.T. J. Biol. Chem. 1987; 262: 3524-3527Abstract Full Text PDF PubMed Google Scholar). The standard inactivation assay was carried out, except 0.1 mm copper sulfate was added to the culture medium 4 h prior to the transfer to glucose and the initiation of the inactivation assay (29Ward A.C. Castelli L.A. MaCreadie I.G. Azad A. Yeast. 1994; 10: 441-449Crossref PubMed Scopus (28) Google Scholar). As can be seen in Fig. 1(bottom two panels), overexpression of ubiquitin in the doa4Δ (pCUP1-Ub) strain suppresses the loss of DOA4, restoring a more rapid rate of glucose-induced proteolysis of maltose permease than that observed in thedoa4Δ (pCUP1-vector) control strain. Table II shows that the steady state transport rate of maltose in a ubiquitin-overexpressing doa4Δ strain, CMY1025 (pCUP1-Ub), is half that seen in the control strain, CMY1025 (pCUP1-vector). This is also consistent with the proposal that ubiquitin is required for rapid turnover of maltose permease. In order to determine whether maltose permease is ubiquitinated directly, we used strain CMY1004, which contains a temperature-sensitive allele of END3 to slow down endocytosis and degradation of maltose permease and thereby enhance the levels of any putative ubiquitinated species (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). END3 is an early function in the endocytosis process (30Raths S. Rohrer J. Crausaz F. Riezman H. J. Cell Biol. 1993; 120: 55-65Crossref PubMed Scopus (319) Google Scholar). We have shown that endocytosis and the subsequent proteolysis of Mal61/HA maltose permease are completely inhibited at the nonpermissive temperature, in end3-ts strains, and that, even at the permissive temperature, maltose permease protein accumulates to higher levels in the plasma membrane (1Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar). Plasmid pCUP1-mycUb (yEP105), which encodes a c-myc-tagged allele of ubiquitin, was introduced into strain CMY1004. Strain CMY1004 (pCUP1-mycUb) was grown to very early log phase (A 600 0.2–0.3) in selective media plus 2% maltose at room temperature, and 0.1 mm CuSO4was added to induce expression of c-myc-Ub (29Ward A.C. Castelli L.A. MaCreadie I.G. Azad A. Yeast. 1994; 10: 441-449Crossref PubMed Scopus (28) Google Scholar). After a 5-h ubiquitin induction period, the culture was transferred to 37 °C to inhibit endocytosis, and then after an additional 1 h glucose was added to a final concentration of 2%. Samples were collected at time points throughout this process, and the level of Mal61/HA protein was determined by Western blotting. Published reports indicate that substrate proteins that are conjugated with a c-myc-tagged allele of ubiquitin are more stable than untagged ubiquitin-substrate protein conjugates and accumulate to a significantly higher level (21Hochstrasser M. Ellison M.J. Chau V. Varshvsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4604-4610Crossref Scopus (197) Google Scholar). As is evident in Fig. 2, overexpression of ubiquitin during the Cu2+ induction period results in the accumulation of a larger molecular weight species of Mal61/HA protein in the end3 strain even prior to the addition of glucose. Thus, it appears that a small amount of a putative ubiquitinated species of maltose permease is present during growth on maltose. The addition of the 2% glucose to the growth medium causes an increase in the abundance of this larger molecular weight band, which peaks at about 30–45 min. In order to confirm that this higher molecular weight species is indeed a ubiquitinated maltose permease, we utilized the modest molecular weight difference produced by conjugation to c-myc-tagged ubiquitin versus untagged ubiquitin. The difference in size between the product encoded by these two alleles, approximately 1.3–1.5 kDa, previously has been used to verify ubiquitinated substrates such as the Matα2 transcriptional regulator (8Galan J.M. Moreau V. Andre B. Volland C. Haguenauer-Tsapis R. J. Biol. Chem. 1996; 271: 10946-10952Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 9Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar, 14Egner R. Kuchler K. FEBS Lett. 1996; 378: 177-181Crossref PubMed Scopus (102) Google Scholar,21Hochstrasser M. Ellison M.J. Chau V. Varshvsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4604-4610Crossref Scopus (197) Google Scholar). Strain CMY1004 (pCUP1-mycUb) expressing the Cu2+-inducible c-myc-tagged ubiquitin and strain CMY1004 (pCUP1-Ub) expressing the Cu2+-inducible untagged ubiquitin were both grown at room temperature to early log phase, and 0.1 mmCuSO4 was added to the growth media. After 4 h, the cultures were moved to 37 °C for 1 h prior to the addition of 2% glucose. After the glucose was added, cells were allowed to continue growing at 37 °C for ½ h and then harvested for Western analysis of Mal61/HAp. As is seen in Fig. 3, both strains carrying the different alleles of ubiquitin exhibit the higher molecular weight species of Mal61/HA protein described above, but in the strain carrying the c-myc-tagged allele of ubiquitin, this species is slightly larger than the corresponding species in the strain carrying the untagged allele of ubiquitin. The c-myc-tagged ubiquitin-maltose permease conjugate also appears to be significantly more abundant than the corresponding untagged species, consistent with reports that the c-mycubiquitin-conjugated proteins are more stable (21Hochstrasser M. Ellison M.J. Chau V. Varshvsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4604-4610Crossref Scopus (197) Google Scholar). These results confirm that this higher molecular weight species is indeed a ubiquitinated maltose permease. The size of this ubiquitinated species of maltose permease is increased by approximately 6–7 kDa, compared with the major species of Mal61/HAp, and is consistent with a monoubiquitinated maltose permease. RSP5/NPI1 encodes a ubiquitin-protein ligase that participates in the induced degradation of at least two permeases: the general amino acid permease, encoded by GAP1, and the uracil permease, encoded by FUR4 (6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar). Anrsp5/npi1 mutant allele was isolated based on its nitrogen repression-resistant phenotype (31Grenson M. Eur. J. Biochem. 1983; 133: 135-139Crossref PubMed Scopus (88) Google Scholar) and has since been shown to be a Ty1 insertion into the RSP5 promoter (6Hein C. Springael J.Y. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (297) Google Scholar). Strains carrying this mutant allele synthesize significantly reduced levels of this essential protein that are adequate for cell growth but insufficient for amm" @default.
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• W2014533300 title "The Role of Ubiquitin Conjugation in Glucose-induced Proteolysis of SaccharomycesMaltose Permease" @default.
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