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• W2106360060 abstract "Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high affinity iron transport system in response to iron need. This transport system is comprised of the products of the FET3 and FTR1 genes. We show that Fet3p and Ftr1p are post-translationally regulated by iron. Incubation of cells in high iron leads to the internalization and degradation of both Fet3p and Ftr1p. Yeast strains defective in endocytosis (Δend4) show a reduced iron-induced loss of Fet3p-Ftr1p. In cells with a deletion in the vacuolar protease PEP4, high iron medium leads to the accumulation of Fet3p and Ftr1p in the vacuole. Iron-induced degradation of Fet3p-Ftr1p is significantly reduced in strains containing a deletion of a gene, VTA1, which is involved in multivesicular body (MVB) sorting in yeast. Sorting through the MVB can involve ubiquitination. We demonstrate that Ftr1p is ubiquitinated, whereas Fet3p is not ubiquitinated. Iron-induced internalization and degradation of Fet3p-Ftr1p occurs in a mutant strain of the E3 ubiquitin ligase RSP5 (rsp5-1), suggesting that Rsp5p is not required. Internalization of Fet3p-Ftr1p is specific for iron and requires both an active Fet3p and Ftr1p, indicating that it is the transport of iron through the iron permease Ftr1p that is responsible for the internalization and degradation of the Fet3p-Ftr1p complex. Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high affinity iron transport system in response to iron need. This transport system is comprised of the products of the FET3 and FTR1 genes. We show that Fet3p and Ftr1p are post-translationally regulated by iron. Incubation of cells in high iron leads to the internalization and degradation of both Fet3p and Ftr1p. Yeast strains defective in endocytosis (Δend4) show a reduced iron-induced loss of Fet3p-Ftr1p. In cells with a deletion in the vacuolar protease PEP4, high iron medium leads to the accumulation of Fet3p and Ftr1p in the vacuole. Iron-induced degradation of Fet3p-Ftr1p is significantly reduced in strains containing a deletion of a gene, VTA1, which is involved in multivesicular body (MVB) sorting in yeast. Sorting through the MVB can involve ubiquitination. We demonstrate that Ftr1p is ubiquitinated, whereas Fet3p is not ubiquitinated. Iron-induced internalization and degradation of Fet3p-Ftr1p occurs in a mutant strain of the E3 ubiquitin ligase RSP5 (rsp5-1), suggesting that Rsp5p is not required. Internalization of Fet3p-Ftr1p is specific for iron and requires both an active Fet3p and Ftr1p, indicating that it is the transport of iron through the iron permease Ftr1p that is responsible for the internalization and degradation of the Fet3p-Ftr1p complex. Transition metals are essential for life, yet transition metals in high concentrations can be toxic. Both eukaryotes and prokaryotes tightly regulate the concentration of free intracellular metals by either regulating metal uptake or sequestration. High affinity iron transport in the budding yeast Saccharomyces cerevisiae requires the expression of two cell surface proteins, the multicopper oxidase Fet3p and the transmembrane permease Ftr1p (1Askwith C.C. de Silva D. Kaplan J. Mol. Microbiol. 1996; 20: 27-34Crossref PubMed Scopus (103) Google Scholar, 2Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (584) Google Scholar). Transcription of these genes, as well as genes that encode proteins required for the processing of Fet3p, is regulated by the iron sensing transcription factor Aft1p (3Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google Scholar). In S. cerevisiae transporters for the transition metals copper and zinc are regulated post-translationally. High levels of zinc induce the internalization and vacuolar degradation of Zrt1p, the high affinity zinc transporter (4Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). High levels of copper induce the degradation of Ctr1p, the high affinity copper transporter, whereas Ctr3p, another high affinity copper transporter, is not affected (5Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (180) Google Scholar). A previous study from our laboratory suggested that regulation of the high affinity iron transport system was predominantly transcriptional (6Eide D. Davis-Kaplan S. Jordan I. Sipe D. Kaplan J. J. Biol. Chem. 1992; 267: 20774-20781Abstract Full Text PDF PubMed Google Scholar), although there is evidence that the activity of the iron transport system may be regulated by cAMP (7Lesuisse E. Horion B. Labbe P. Hilger F. Biochem. J. 1991; 280: 545-548Crossref PubMed Scopus (29) Google Scholar). Studies in Schizosaccharomyces pombe, however, suggested that the multicopper oxidase-based high affinity iron transport system might be regulated post-translationally. Incubation of S. pombe, expressing the high affinity transport system, with high concentrations of iron led to a rapid inhibition of iron transport (8Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). High levels of iron transport activity are seen when FET3/FTR1 are expressed using the iron-independent GAL10 promoter. There is a 50% reduction in transport activity when such cells are incubated in high iron as opposed to low iron medium (8Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Based on these observations, we re-examined whether the Fet3p-Ftr1p transport system is post-translationally regulated. We demonstrate that high levels of iron induce the internalization and degradation of the Fet3p-Ftr1p transport system. Strains and Media—The S. cerevisiae strains used in this study are listed in Table I. The cells were grown in either medium containing yeast extract-peptone-dextrose (YPD) 1The abbreviations used are: YPD, yeast extract-peptone-dextrose; BPS, bathophenanthroline disulfonate; CM, complete media; GFP, green fluorescent protein; CFP, cyan fluorescent protein. or yeast nitrogen base synthetic complete medium (CM) with supplements as needed (9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar). Low iron growth medium was made by adding 40 or 80 μm bathophenanthroline disulfonate (BPS), an iron chelator, to CM or YPD and then adding back varying amounts of FeSO4 (9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar). Low iron medium used in this work is referred to as BPS (x), in which the media contains BPS and x equals the concentration in micromolar of added FeSO4.Table IStrains used in this studyStrainGenotypeRef.DY150MATa ade2, can1, his3, leu2, trp1, ura3(9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar)DY1457MATα ade6, can1, his3, leu2, trp1, ura3(9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar)DY150 (FET3-GFP)DY150, FET3-GFP::KanMXThis studyDY1457 (FTR1-CFP)DY1457, FTR1-CFP::KanMXThis studyΔend4DY150, Δend4::LEU2(37Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar)Δftr1 (42C)MAT a ade2, his3, leu2, lys2, trp1, Δftr1::TRP1This studyΔftr1 42C (FET3-GFP)Δftr1 (42C) FET3GFP::KanMX(38Spizzo T. Byersdorfer C. Duesterhoeft S. Eide D. Mol. Gen. Genet. 1997; 256: 547-556PubMed Google Scholar)Δpep4MATa ade2, can1, his3, leu2, trp1, ura3, Δpep4::URA3This studyΔpep4Δfet3MATa ade2, can1, his3, leu2, trp1, ura3, Δpep4::URA3, Δfet3:KanMXThis studyΔgef1MATα ade6, can1, his3, leu2, trp1, ura3(14Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar)BY4742MATα his3 leu2 lys2 ura3(21Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)Δvta1-5aMATα his3 leu2 met 15 ura3, Δvta1::KanMX(21Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)OCY 354MATa ade2, can1, his3, leu2, trp1, ura3, HO::FET3 LacZThis study23344MATα, ura3(15Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar)27038 (rsp5)MATa, ura3, rsp5(15Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar)23344 (FET3-GFP)MATα, ura3, FET3-GFP::KanMXThis study27038 (rsp5) (FET3-GFP)MATa, ura3, rsp5, FET3-GFP::KanMXThis study23344 (FTR1-CFP)MATα, ura3, FTR1-CFP::KanMXThis study27038 (rsp5) (FTR1-CFP)MATa, ura3, rsp5, FTR1-CFP::KanMXThis studyLHY291His3, trp1, ade2, ura3, leu2, bar1(24Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (120) Google Scholar)LHY23rsp5-1, ura3, leu2, trp1, bar1 GAL(24Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (120) Google Scholar) Open table in a new tab S1 Nuclease Protection Analysis—Total RNA was isolated and analyzed using standard techniques (10Chen O.S. Hemenway S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16922-16927Crossref PubMed Scopus (38) Google Scholar). All samples were isolated from mid-log phase cultures grown in either CM or CM BPS (5Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (180) Google Scholar). The 32P-labeled FET3 and CMD1 probes were generated. Preparation of Antisera against Fet3p—A secreted Fet3p (Fet3p lacking the transmembrane and cytoplasmic domains) was generated as described by Hasset et al. (11Hassett R.F. Yuan D.S. Kosman D.J. J. Biol. Chem. 1998; 273: 23274-23282Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Procedures for the isolation and deglycosylation of secreted-Fet3p have been described previously (12Harris Z.L. Davis-Kaplan S.R. Gitlin J.D. Kaplan J. Blood. 2004; 103: 4672-4673Crossref PubMed Scopus (28) Google Scholar). The N-glycanase-treated Fet3p was injected into rabbits, and antisera were prepared. The soluble Fet3p was attached to an Amino-link gel using the manufacturer's instructions (Pierce Inc.). The antiserum was applied to the column, and the column was extensively washed with phosphate-buffered saline and eluted with 0.1 m glycine (pH 2.5) and immediately neutralized with 1.0 m Tris-HCl (pH 9.0). The purified antibody was useful for both immunofluorescence and Western analysis. Immunofluorescence—Cells were prepared for immunofluorescence as described previously (13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For visualization of Fet3p, the rabbit anti-Fet3p antibody was used (1:500) followed by either an Alexa 594- or Alexa 488-conjugated goat anti-rabbit antibody (1:500). All of the fluorescent secondary antibodies were obtained from Molecular Probes. Western Analysis—Western blot analysis was performed on Fet3p-containing membrane fractions as described previously (14Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar) using our purified rabbit anti-Fet3p (1:1000). The only variation in protocol was that membranes (15 μg) were treated with endoglycosidase Hf per the manufacturer's protocol (New England Biolabs) before being analyzed by SDS-PAGE using 10% gels followed by Western analysis. For Western analysis of Gap1p-GFP or Ftr1p-CFP, membranes were isolated using a procedure described previously (15Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar) and analyzed on 10% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were blocked with milk and incubated with either a rabbit anti-Fet3p (1:1,000), rabbit anti-GFP (1:10,000) (Abcam #6556), or rabbit anti-Gas1p (1:30,000, the kind gift of Dr. Howard Riezman University of Basel), followed by peroxidase-conjugated goat anti-rabbit IgG (1:12,500, Jackson ImmunoResearch Laboratories). Proteins were visualized by using a Western Lightning chemiluminescent detection system (PerkinElmer Life Sciences). Immunoprecipitation—For immunoprecipitation of Fet3p-GFP, Gap1p-GFP, or Ftr1p-CFP, proteins were extracted as described for Western analysis. Following extractions, samples were incubated with rabbit anti-GFP antibody (1:10,000, Abcam #6556) and 40 μl of Sepharose-conjugated Protein A/G beads (Santa Cruz Biotechnology) overnight at 4 °C. Protein A/G beads were pelleted and washed ten times in extraction/lysis buffer, and samples were eluted in 2× SDS-PAGE sample buffer without β-mercaptoethanol. Immunoprecipitated samples were examined by SDS-PAGE followed by Western analysis using either rabbit anti-Fet3p, mouse anti-GFP (1:10,000, Covance), or mouse anti-ubiquitin (1:1,000, Covance) as the primary antibody and peroxidase-conjugated goat anti-mouse or rabbit IgG as the secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories, Inc.). Atomic Absorption Assay—Cells were grown to log phase in low iron medium and then transferred to medium containing a range of FeSO4 for 2 h. Log phase cells were collected and washed by centrifugation with 50 mm Tris-HCl, pH 6.5, 10 mm EDTA. Cell pellets were digested in 200 μl of 5:2 nitric acid:perchloric acid at 80 °C for 1 h. After digestion, the samples were diluted to 1.0 ml with deionized water and then flamed in a PerkinElmer Life Sciences inductively coupled plasma atomic absorption spectrometer. All samples were measured in duplicate, and the experiment was performed at least twice. To determine if the Fet3p-Ftr1p transport system is post-transcriptionally regulated by iron, we exposed wild type cells expressing the transport system to high iron medium and then examined Fet3p levels by Western analysis. When cells were exposed to high iron medium there was a concentration-dependent decrease in Fet3p. Relative to Gas1p, employed as a loading control, exposure of cells to 1 mm FeSO4 resulted in the disappearance of 50% of Fet3p within 1 h (data not shown) and 80% within 2 h (Fig. 1A). It may be possible that the disappearance of surface Fet3p is the result of the steady-state turnover of Fet3p, as FET3 transcription is iron-sensitive (9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar). We observed that FET3 mRNA levels were dramatically decreased when cells were incubated with as little as 10 μm iron (Fig. 1B). There was little further change in transcript level with increased medium iron. Examination of Fet3p levels revealed little decrement in Fet3p when cells were incubated with 10 μm iron for 2 h. Decreased protein levels were only seen at higher concentrations of iron (Fig. 1A). These results suggest that Fet3p levels may be regulated independently of FET3 mRNA. We confirmed this result using two different approaches. First, we measured Fet3 protein in cells treated with the protein synthesis inhibitor cycloheximide. Cells were grown in low iron medium, and cycloheximide was added at the same time as high iron. In the presence of cycloheximide, there was an iron-dependent decrease in Fet3p (Fig. 1C). Second, we examined changes in Fet3p levels in Δfet3 cells transformed with a plasmid containing GAL10FET3 (8Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). High affinity iron transport occurs under low iron conditions in the presence of galactose but not in the presence of glucose. Cells were exposed to galactose in low iron medium to induce the high affinity iron transport system. The cells were then incubated in glucose media to prevent transcription of FET3 mRNA. Addition of glucose leads to inhibition of transcription of galactose-regulated genes within 4 min (16Mason P.B. Struhl K. Mol. Cell. Biol. 2003; 23: 8323-8333Crossref PubMed Scopus (269) Google Scholar). Addition of iron to glucose media resulted in a concentration-dependent loss of Fet3p (Fig. 1D). These experiments demonstrate that iron has an effect on Fet3p independent of FET3 transcription. The post-translational regulation of the high affinity iron transport system was confirmed using immunofluorescence. Cells incubated in iron-depleted medium showed fluorescent staining of the cell surface, whereas Δfet3 cells stained with the same anti-Fet3p antibody showed no fluorescence (Fig. 2A). Addition of iron for 2 h resulted in the disappearance of Fet3p fluorescence. Expression of FET3 regulated by the GAL10 promoter leads to abundant Fet3p on the cell surface, and there was little change in the surface expression of Fet3p when galactose-grown cells were incubated in glucose-containing medium. Upon addition of iron, there was a dramatic decrease in fluorescence (data not shown). We generated strains containing an integrated FET3-GFP. As shown previously, addition of an epitope to either Fet3p or Ftr1p does not alter their ability to transport iron (17Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar). Addition of iron also resulted in the loss of surface fluorescence in cells that had a chromosomal copy of FET3 with a carboxyl-terminal GFP (Fig. 2B). The loss of Fet3p was confirmed by Western analysis. Both components of the high affinity iron transport system, Fet3p and Ftr1p, have to be synthesized simultaneously for appropriate cell surface targeting (2Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (584) Google Scholar). In the absence of Fet3p, Ftr1p does not localize to the surface and is degraded, as is Fet3p in the absence of Ftr1p. These results suggest that Fet3p and Ftr1p form a complex. Based on the observation that iron induced the loss of Fet3p, we asked whether iron also induced the loss of Ftr1p. We generated strains containing an integrated FTR1-CFP, because published studies show the utility of this fusion protein (17Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar). Expression of Frt1p-CFP permitted those cells to grow on low iron media, indicating that the protein was functional. When Ftr1p-CFP-expressing cells were incubated with high iron there was a loss of cell surface Ftr1p-CFP fluorescence (Fig. 2C). The predicted molecular mass of Ftr1p is 45.7 kDa, and addition of CFP would add 27 kDa. Our data show Ftr1p-CFP migrating on SDS-PAGE with a predicted molecular mass of 70 kDa, which is close to the predicted size of the fusion protein. We took advantage of mutant cell lines to show that the loss of surface Fet3p was due to internalization and vacuolar degradation. A deletion of END4 attenuates but does not completely inhibit endocytosis, as shown by decreased uptake of the fluorescent dye FM4–64 (18Raths S. Rohrer J. Crausaz F. Riezman H. J. Cell Biol. 1993; 120: 55-65Crossref PubMed Scopus (319) Google Scholar). In Δend4 cells, the iron-induced loss of surface Fet3p was reduced (Fig. 3A). In cells that lack the vacuolar protease Pep4p, the iron-induced loss of cell surface fluorescence correlated with the appearance of fluorescence in the vacuole (Fig. 3B). The targeting of many cell surface proteins to the vacuoles requires their sorting in the multivesicular body (19Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1026) Google Scholar). Vta1p is a class E protein involved in multivesicular body sorting (20Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar, 21Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In iron-exposed Δvta1 cells, Fet3p was not degraded and was found at the plasma membrane as well as in a class E prevacuolar compartment (Fig. 3C). The Fet3p-Ftr1p transport system is highly specific for iron and will not transport other transition metals. Cells with a deletion of PEP4 (Δpep4) were incubated overnight in low iron medium and then exposed to different transition metals. At a concentration of 50 μm, only iron led to the vacuolar accumulation of Fet3p. Incubation of cells in metals such as Cu+, Mn2+, and Zn2+ in concentrations as high as 50 μm did not lead to internalization of Fet3p (Fig. 4). These results show that internalization of surface Fet3p is specific for iron. Entry of most plasma membrane proteins into the vacuole through the multivesicular body is a consequence of their being ubiquitinated. We therefore examined whether either Fet3p or Ftr1p was ubiquitinated. Cells expressing either Fet3p-GFP or Ftr1p-CFP were incubated in the presence of iron for 2 h and harvested, and detergent extracts were immunoprecipitated with anti-GFP antibodies. Western blots of the immunoprecipitates were probed with an anti-ubiquitin antibody. No ubiquitin was seen in the immunoprecipitate from Fet3p-GFP-expressing cells, but ubiquitin was found in extracts from Ftr1p-CFP-expressing cells (Fig. 5A). Ubiquitin could be seen on Ftr1p-CFP from cells grown in low iron medium; however, the addition of iron resulted in a significant increase in ubiquitin levels. Immunoprecipitated Ftr1p (detected by Western analysis) had an apparent molecular mass of 45 kDa, much lower than that of Ftr1p-CFP seen in extracts of cells probed by Western analysis. We think that it is likely that the lower molecular mass results from proteolytic cleavage occurring during immunoprecipitation, because we did not observe this change in molecular mass prior to immunoprecipitation (compare Figs. 2B and 5A). We confirmed the presence of ubiquitin on Ftr1p by taking advantage of cells transformed with a plasmid containing a copper regulated (CUP1) ubiquitin with a carboxyl-terminal c-myc epitope. Cells were grown in high copper-containing medium to induce the expression of ubiquitin-c-myc, and detergent extracts were immunoprecipitated using antibodies to GFP. Again, no ubiquitin was seen in immunoprecipitates from Fet3p-GFP cells, but ubiquitin was seen in immunoprecipitates from Ftr1p-CFP cells (Fig. 5B). We observed that Ftr1p was ubiquitinated in both low and high iron medium with multiple ubiquitin-containing bands. Other plasma membrane proteins have been found to be hyper-ubiquitinated in cells overexpressing ubiquitin (15Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar). If ubiquitination of Ftr1p was responsible for the internalization of the Fet3p-Ftr1p complex, then we might expect increased internalization of hyper-ubiquitinated Ftr1p-CFP in low iron medium. In Fet3p-GFP- or Ftr1p-CFP-expressing cells incubated in low iron medium, fluorescence was found predominately on the cell surface. Under the same conditions, in cells expressing ubiquitin, fluorescence was now found in the vacuole. Addition of iron to cells overexpressing ubiquitin resulted in an increase in the rate of vacuolar accumulation of Fet3p-Ftr1p. These results suggest that ubiquitination of Ftr1p leads to internalization of the Fet3p-Ftr1p complex. Most plasma membrane transporters are ubiquitinated by the ubiquitin ligase Rsp5p (for reviews see Refs. 19Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1026) Google Scholar and 22Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (967) Google Scholar). To determine if Rsp5p is required for the iron-induced internalization of Fet3p-Ftr1p transport system, we utilized a yeast strain with a mutation in RSP5. We first confirmed that rsp5 cells showed a defect in ubiquitination by following the degradation of the high affinity amino acid transporter Gap1p. Wild type and rsp5 cells were transformed with a GAP1-GFP plasmid. In cells grown in ammonia-free (nitrogen-poor) media Gap1p is localized to the plasma membrane (15Springael J.Y. Andre B. Mol. Biol. Cell. 1998; 9: 1253-1263Crossref PubMed Scopus (186) Google Scholar, 23Soetens O. De Craene J.O. Andre B. J. Biol. Chem. 2001; 276: 43949-43957Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Addition of ammonia results in the internalization of Gap1p-GFP and its localization in the vacuole, as seen by fluorescence or by the presence of cleaved GFP on Western blots. In the rsp5 cells, Gap1p-GFP is found at the plasma membrane and in a prevacuolar compartment (Fig. 6A). Western analysis of rsp5 extracts showed full-length Gap1p-GFP, whereas in wild type cells only GFP is seen, indicating that Gap1-GFP is degraded. Gitan and Eide (4Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) reported a decrease in the Zn2+-mediated internalization and degradation of the high affinity zinc transporter Zrt1p in rsp5 cells. We confirmed that result, as shown in Fig. 6B. To determine if Rsp5 was necessary for the iron-induced degradation of Fet3p-Ftr1p, we generated an rsp5 strain with an integrated FET3-GFP or FTR1-CFP. We were surprised to find no reduction in iron-induced internalization or degradation of Fet3p or Ftr1p in rsp5 cells (Fig. 6C). It is possible that the rsp5 mutant retains sufficient activity to ubiquitinate Ftr1p. We therefore took advantage of a temperature-sensitive allele of RSP5 (rsp5-1), which shows a severe defect in ubiquitination (24Dunn R. Hicke L. Mol. Biol. Cell. 2001; 12: 421-435Crossref PubMed Scopus (120) Google Scholar). We confirmed that rsp5-1 has a temperature-sensitive defect in ubiquitination by showing a severe alteration in α-factor-mediated internalization of Ste2p-GFP (Fig. 6D). Fet3p-GFP showed no of iron-dependent loss in rsp5-1 cells at the restrictive temperature (Fig. 6E). These results suggest that Rsp5p is not required for the iron-induced loss of the high affinity iron transport system. We considered three mechanisms to explain how iron signals the internalization and degradation of Fet3p-Ftr1p: a signal generated by iron at the cell surface, a signal generated by iron inside the cell, or a signal generated as a consequence of movement of iron through the transport system. To test these possibilities, we transformed a Δfet3Δpep4 strain with a GAL10-regulated allele of FET3 that was unable to transport iron due to a mutation in one of the amino acids that ligate the Type 1 copper (25Askwith C.C. Kaplan J. J. Biol. Chem. 1998; 273: 22415-22419Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cells were grown in galactose to induce the expression of the mutant Fet3p. Although the inactive Fet3p is still translocated to the cell surface, incubation of cells with high levels of iron did not result in the degradation of the mutant Fet3p, as assessed by either Western blot analysis (data not shown) or by immunofluorescence (Fig. 7A). We then examined the effect of mutations in Ftr1p by expressing a form of Ftr1p that had defective iron transport. A mutation in the putative iron binding REXLE domain of Ftr1p does not prevent Ftr1p and Fet3p from being localized to the cell surface but reduces iron transport by ∼80% (17Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar). When exposed to high iron, the degradation of the transport system was reduced compared with wild type cells (Fig. 7B). The concentration of media iron in these experiments was high, because it was sufficient to support the growth of Δfet3 cells but was also sufficient to inhibit transcription of a FET3lacZ reporter construct in Δfet3 cells (data not shown). To further show that a Fet3-Ftr1p transport system is required for iron-induced internalization, we took advantage of cells with a deletion in the GEF1 gene. Gef1p is a voltage-regulated chloride channel present in the post-Golgi compartment in which apo-Fet3p is copper-loaded (14Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar, 26Gaxiola R.A. Yuan D.S. Klausner R.D. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4046-4050Crossref PubMed Scopus (152) Google Scholar). In the absence of Gef1p, apo-Fet3p is not copper-loaded but is still targeted to the cell surface. Cell surface apo-Fet3p, lacks multicopper oxidase activity and is unable to transport iron. Incubation of Δgef1 cells with iron did not lead to the degradation of apo-Fet3p (Fig. 8). Apo-Fet3p on the cell surface can be copper-loaded by incubation of cells at 0 °C in the presence of Cl–,Cu+, and reduced pH (14Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar). Copper loading of apo-Fet3p resulted in increased iron transport activity and increased multicopper oxidase activity. Once copper-loaded, addition of iron leads to the internalization and degradation of Fet3p. These results demonstrate that an active iron transport system is required for iron to induce the internalization of Fet3p-Ftr1p. Extracellular iron is not the signal for the internalization and degradation of the high affinity iron transport system. S. cerevisiae can acquire iron through the low affinity iron transport system, Fet4p, as well as through siderophore-iron transporters. There are two separate routes by which S. cerevisiae can acquire iron provided by iron-siderophore complexes (27Lesuisse E. Simon-Casteras M. Labbe P. Microbiology. 1998; 144: 3455-3462Crossref PubMed Scopus (130) Google Scholar, 28Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The first route involves reduction of siderophore-iron complexes at the cell surface followed by the uptake of iron by the Fet3p-Ftr1p transport system. The impermeable Fe(II) chelator BPS can inhibit uptake of iron by this route. The second route of uptake of siderophore iron involves transport of the siderophore iron complex through a siderophore transporter. This route of iron acquisition cannot be inhibited by BPS. In the presence of high concentrations of BPS, yeast can grow on siderophore-iron complexes (28Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 29Kosman D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (265) Google Scholar). Addition of high concentrations of ferrioxamine-iron to cells grown in BPS did not lead to the internalization of Fet3p-Ftr1p (Fig. 9A). The same concentration of ferrioxamine-iron, however, can provide enough iron to support the growth of cells and to prevent the expression of a FET3lacZ reporter construct (Fig. 9B). It may be possible that the amount of siderophore-iron accumulated within cells may be enough to suppress the transcription of the iron-regulon but is insufficient to induce the internalization of Fet3p-Ftr1p. To examine the effect of intracellular iron on the degradation of Fet3p-Ftr1p, we took advantage of the observation that, in the absence of high affinity, iron transport system cells increase the expression of the low affinity transition metal transporter Fet4p (30Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). As shown above, Δgef1 cells do not internalize Fet3p-Ftr1p. At low concentrations of media iron wild type cells accumulated more iron than Δgef1 cells (Fig. 9C). This is expected, because Δgef1 cells do not have a functional high affinity iron transport system. As media iron increases, Δgef1 cells showed a greater accumulation of iron than wild type cells. Increased iron accumulation reflects the increased expression of the Fet4p low affinity iron transporter on Δgef1 cells and low levels of Fet4p on wild type cells. Even in the face of greater than wild type levels of cellular iron, no degradation of Fet3p-Ftr1p was observed (see Fig. 8). These results suggest that intracellular iron does not provide the signal for the internalization of Fet3p-Ftr1p. Transcriptional regulation of the high affinity iron transport system, comprising the products of the FET3 and FTR1 genes, by the transcription factor Aft1p has been well described (3Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google Scholar, 31Casas C. Aldea M. Espinet C. Gallego C. Gil R. Herrero E. Yeast. 1997; 13: 621-637Crossref PubMed Scopus (76) Google Scholar, 32Rutherford J.C. Jaron S. Winge D.R. J. Biol. Chem. 2003; 278: 27636-27643Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (292) Google Scholar). We now demonstrate that Fet3p and Ftr1p are regulated post-translationally, as iron induces the internalization and degradation of both Fet3p and Ftr1p. The simultaneous synthesis of Fet3p and Ftr1p is required for their appropriate targeting to the cell surface suggesting that these molecules are in a complex (2Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (584) Google Scholar, 8Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The observation that iron induces the simultaneous internalization of both Fet3p and Ftr1p provides further support for the view that these two proteins exist in an obligate complex. Iron-induced internalization of the iron transport system is consistent with studies showing post-translational regulation of copper (5Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (180) Google Scholar), zinc (4Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 34Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar), and manganese (35Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) transport systems. For both iron and zinc transport systems, transcriptional regulation is more sensitive than post-translational regulation. Iron, at concentrations as low as 10 μm, can reduce transcription of FET3 by >90%, whereas the same concentration only had minimal effects on protein levels. Ubiquitination is the signal that targets most membrane proteins for degradation (19Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1026) Google Scholar, 22Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (967) Google Scholar). Our data indicate that Ftr1p can be ubiquitinated and that ubiquitination is required for degradation, because deletions of genes required for sorting into the multivesicular body pathway prevent the vacuolar localization of Fet3p-Ftr1p. Most plasma membrane proteins are ubiquitinated by Rsp5p (22Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (967) Google Scholar). Rsp5p, in combination with Bsd2p, is responsible for ubiquitination of the Mn2+ transporter Smf1p in the biosynthetic pathway (36Hettema E.H. Valdez-Taubas J. Pelham H.R. EMBO J. 2004; 23: 1279-1288Crossref PubMed Scopus (128) Google Scholar). The rate of degradation of the amino acid transporter Gap1p and the zinc transporter Zrt1p was severely reduced in rsp5 cells. It was surprising to find that the iron-induced degradation of Fet3p-Ftr1p was not affected in rsp5 cells. Furthermore, no iron-induced change in surface Fet3p was seen at the restrictive temperature in cells that had temperature-sensitive allele of RSP5, although effects were seen on the internalization of Ste2p. These results suggest that a ubiquitin ligase other than Rsp5p is required for the ubiquitination of Ftr1p. Given that metals can induce the internalization of surface transporters via ubiquitination leading to transporter degradation, what is the signal that leads to ubiquitination? The metal-induced event that leads to Rsp5p-mediated ubiquitin addition in Smf1p may be a conformational change in the transporter resulting from transport of the metal (36Hettema E.H. Valdez-Taubas J. Pelham H.R. EMBO J. 2004; 23: 1279-1288Crossref PubMed Scopus (128) Google Scholar). Mutations that abolish transport activity for Smf1p abolish metal-induced internalization. These studies suggest that Rsp5p (in combination with Bsd2p) recognizes alterations in the hydrophobic domain of membrane proteins. Transport of substrate might be expected to lead to perturbation in the lipid bilayer resulting from movements in the transporter as substrate is passed through the bilayer. This is an attractive model for post-translational regulation of the iron transport system, because it would correlate transport activity with both cellular metal requirement and transcriptional regulation of the high affinity iron transporter. In conditions of iron sufficiency, iron transport will lead to degradation of the transporter. In the face of iron insufficiency, even though transport of iron increases the rate of degradation of the transporter, the increased degradation rate will be offset by increased transcription of the transporter. Linking transport activity to degradation rate provides a simple feedback mechanism that ensures tight control of cytosolic metal levels, as well as assuring the specificity of membrane targeting to the specific transporter. The demonstration that the Fet3p-Ftr1p transport system must be active to effect iron-induced internalization indicates that cell surface iron is not the signal for internalization/ubiquitination. There are two issues that must be resolved before accepting a model for iron-induced internalization in which the movement of iron through the channel is responsible for ubiquitination. First, why are high concentrations of iron required for post-translational regulation? The Km for the Fet3p-Ftr1p transport system is in the sub-micromolar (0.15–0.2 μm) range (9Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (590) Google Scholar), yet much higher concentrations of iron (>50 μm) are required for substantial rates of transporter degradation. Second, what is the ubiquitin ligase responsible for “marking” Ftr1p? We have ruled out Rsp5p, although it is formally possible that, even in the rsp5 mutant strain or the temperature-sensitive rsp5-1 strain, residual enzyme activity is sufficient to ubiquitinate Ftr1p. The best way to show that Rsp5p is not required is to identify the ubiquitin ligase that is required. Those studies are in progress. We thank Drs. David Eide (University of Wisconsin), Chris Kaiser (Massachusetts Institute of Technology), Linda Hicke (Northwestern), and Howard Riezman (University of Basel) for their generous gifts of plasmids, cells, and antibodies." @default.
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