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• W1966642383 abstract "The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the ∼13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p·Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM. The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the ∼13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p·Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM. The high affinity iron uptake complex in the plasma membrane of Saccharomyces cerevisiae consists of two proteins at least (1Kosman D.J. Adv. Protein Chem. 2002; 60: 221-269Crossref PubMed Scopus (37) Google Scholar, 2Kosman D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (259) Google Scholar, 3Van Ho A. Ward D.M. Kaplan J. Annu. Rev. Microbiol. 2002; 56: 237-261Crossref PubMed Scopus (179) Google Scholar, 4Stoj C.S. Kosman D.J. King R.B. 2nd Ed. Encyclopedia of Inorganic Chemistry. II. John Wiley & Sons, Inc., New York2005: 1134-1159Google Scholar). Fet3p is a multicopper oxidase (MCO) 2The abbreviations used are: MCO, multicopper oxidase; TM, transmembrane; PM, plasma membrane; FRET, fluorescence resonance energy transfer; UPR, unfolded protein response; GFP/YFP/CFP, green/yellow/cyan fluorescent protein; ER, endoplasmic reticulum; IP, immunoprecipitation; DBD, DNA binding domain; CT, carboxyl-terminal. that catalyzes the oxidation of Fe(II) to Fe(III) using O2 as substrate in what is known as the ferroxidase reaction (5Frieden E. Osaki S. Adv. Exp. Med. Biol. 1974; 48: 235-265Crossref PubMed Scopus (27) Google Scholar). The Fe(III) generated by Fet3p is ligand for the iron permease, Ftr1p (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar). Fet3p is a type I membrane protein with an orientation that places the amino-terminal oxidase domain in the exocellular space (Nexo) and the carboxyl terminus in the cytoplasm (Ccyt) (1Kosman D.J. Adv. Protein Chem. 2002; 60: 221-269Crossref PubMed Scopus (37) Google Scholar, 7de Silva D.M. Askwith C.C. Eide D. Kaplan J. J. Biol. Chem. 1995; 270: 1098-1101Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Ftr1p, which has seven transmembrane (TM) domains, has an identical orientation, Nexo/Ccyt (8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar). Iron uptake through this complex is thought to follow the sequence Fe(II) oxidation by Fet3p, Fe(III) trafficking to Ftr1p, and Fe(III) permeation through Ftr1p into the cytoplasm. Amino acid residues on both Fet3p and Ftr1p have been identified that are essential to one or more of these steps in iron uptake (8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar, 9Bonaccorsi di Patti M.C. Felice M.R. Camuti A.P. Lania A. Musci G. FEBS Lett. 2000; 472: 283-286Crossref PubMed Scopus (30) Google Scholar, 10Quintanar L. Gebhard M. Wang T.-P. Kosman D.J. Solomon E.I. J. Am. Chem. Soc. 2004; 126: 6579-6589Crossref PubMed Scopus (60) Google Scholar, 11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar). This model requires that Fet3p and Ftr1p are in close proximity in the plasma membrane, if not in an actual protein complex. Data do indicate that these two proteins are part of a hetero-oligomeric complex, either a dimer or higher order structure. The most compelling evidence for this complex is the fact that trafficking of either protein to the plasma membrane (PM) requires the presence of the other (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar, 8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar, 11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar). For example, Fet3p produced in an ftr1Δ-containing yeast strain remains in perinuclear and vesicular compartments (8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar, 11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar). This interdependence in trafficking for both proteins has been vividly demonstrated using fluorescent fusions of Fet3p and Ftr1p. These fusion proteins exhibit wild type trafficking and iron uptake characteristics, making them excellent reporters for the former process (8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar, 11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar). There are no data that directly show an interaction between the two proteins e.g. via a yeast two-hybrid analysis. In contrast, interaction trap technology has shown an interaction between the Fet3p·Ftr1p paralogs in S. cerevisiae, Fet5p and Fth1p (12Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3998) Google Scholar, 13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). These two proteins form an iron permease complex in the membrane of the yeast vacuole. The orientation of this complex is equivalent to that of the Fet3p·Ftr1p one (Nlum/Ccyt); therefore, the Fet5p·Fth1p complex most likely transports iron out of the vacuole into the cytoplasm (13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). FET5 was isolated as a high copy suppressor of the iron-uptake (respiratory) deficiency of a fet3Δ-containing strain indicating that Fet5p could functionally replace Fet3p in the Fet3p·Ftr1p complex. However, this complementation, although sufficient to support iron uptake, was kinetically limited, suggesting that Fet5p·Ftr1p complex formation, trafficking, and/or activity was driven by the overproduction of Fet5p and not by a native-like interaction between the two proteins (14Spizzo T. Byersdorfer C. Duesterhoeft S. Eide D. Mol. Gen. Genet. 1997; 256: 547-556PubMed Google Scholar). A similar specificity in ferroxidase permease protein partners is indicated also by the behavior of the Schizosaccharomyces pombe Fio1 and Fip1 proteins (ferroxidase and permease, respectively) when produced in S. cerevisiae. Although simultaneous expression of both S. pombe genes, fio1+and fip1+, nicely restored iron uptake in a fet3Δ-containing S. cerevisiae strain, expression of fio1+alone did not (15Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In other words, although the S. pombe proteins were processed and trafficked normally in S. cerevisiae, the S. pombe ferroxidase, Fio1, cannot assemble with Ftr1p, even when the former protein is overproduced. In addition to its interaction with Ftr1p, Fet3p undergoes two other post-translational modifications; it is strongly glycosylated, and it must acquire its four prosthetic group copper atoms characteristic of all MCO proteins (4Stoj C.S. Kosman D.J. King R.B. 2nd Ed. Encyclopedia of Inorganic Chemistry. II. John Wiley & Sons, Inc., New York2005: 1134-1159Google Scholar). The relationships between these three post-translational events and between them and the trafficking of the proteins to the plasma membrane have not been fully elucidated, although some key aspects have been established. First, the Fet3p produced in the absence of Ftr1p does not acquire its copper atoms but remains in an apo form mostly confined to perinuclear compartments (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar, 8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar, 11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar, 16Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Davis-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). In comparison, Fet3p produced in the presence of Ftr1p but in a copper-deficient cell or in a cell carrying a defective CCC2 gene is found at the plasma membrane, again in its apo form (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar, 16Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Davis-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, 18Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (393) Google Scholar). In other words Ftr1p is required for normal trafficking, but copper acquisition is not. Corresponding to this behavior is the normal membrane localization of a Fet3p protein engineered to lack one of its four copper atoms, Fet3p(T1D) (19Shi X. Stoj C. Romeo A. Kosman D.J. Zhu Z. J. Biol. Chem. 2003; 278: 50309-50315Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). With respect to CCC2, this gene encodes a P-type Cu-ATPase that likely pumps copper into a trans- or post-Golgi compartment and thereby supplies the copper for Fet3p activation (16Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 18Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (393) Google Scholar). There is no firm evidence that complex assembly, trafficking out of the perinuclear compartment(s), or copper loading is linked to Fet3p glycosylation or vice versa. apoFet3p retained in the endoplasmic reticulum (ER) in an ftr1Δ-containing strain is still glycosylated, although only by the addition of the (GlcNAc)2-Man(Man) core to the protein 13 likely N-linked glycosylation sites of the protein (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar, 20Taylor A.B. Stoj C.S. Ziegler L. Kosman D.J. Hart P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15459-15464Crossref PubMed Scopus (169) Google Scholar, 21Sato M. Sato K. Nakano A. Mol. Biol. Cell. 2004; 15: 1417-1424Crossref PubMed Scopus (41) Google Scholar). As part of our systematic analyses of the structure-function relationships in Fet3p and Ftr1p, we wished to explore the structural features of these two proteins that were required for their assembly into an activation- and trafficking-competent complex. We first combined two-hybrid analysis with confocal fluorescence microscopy to correlate the Fet3p·Ftr1p interaction with the cell locale of each protein or of the protein complex. Using fluorescence resonance energy transfer (FRET) we demonstrated also that the two proteins were adjacent to each other if not in a specific complex. In these analyses, we used a number of mutant forms of both proteins to establish a pattern of structure-function with respect to the interactions within and trafficking of the Fet3p·Ftr1p complex. This pattern was then correlated to the copper activation of the apoFet3p as indicated by oxidase assay and the ability of the protein to support iron uptake. Strains, Media, and Culture Conditions—The strain used in the majority of the studies described was AJS05, which was derived from DEY1457 (MATα can1 his3 leu2 trp1 ura3 ade6) (22Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The AJS05 genotype is MATα can1 his3 leu2 trp1 ura3 ade6 fet3::HIS3 ftr1::TRP1 aft1::AFT1-1upKAN. The AFT1-1up allele codes for a constitutively active form of the Aft1p transcription factor that drives expression of the FET3 (and FTR1) locus (23Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar). In this background, expression of episomally expressed wild type and mutant alleles of FET3 and FTR1 and of the chimeric genes was maximized affording cultures that contained the protein partners in the plasma membrane as described. Briefly, AJS05 was constructed by first amplifying the AFT1 gene (+93 to +2570) containing the C591F Aft1-1up mutation from vector pT14 (24Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar). The fragment was cloned into the multiple cloning site in pFA KanMX (25Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2237) Google Scholar). BamHI, which cleaves AFT1 at base pair 306, was used to linearize the plasmid. The linear fragment was integrated in the yeast strain DEY1457fet3Δftr1Δ; integrants were selected on G418-containing plates. Genomic DNA was isolated from the transformants, and integration was confirmed by PCR. The Aft1-1up phenotype was confirmed by determining the iron uptake in transformants in the presence or absence of 500 μm ferrozine. A clone showing similar iron uptake in the presence or absence of iron chelator was selected for further experiments. To assess the retrieval of Fet3 species from the plasma membrane to the vacuole, a fet3Δend3Δ-containing strain was used that was derived from the END3 knock-out strain, YO2992 (Open Biosystems, Huntsville, AL). The genotype of this strain was MATa his3 leu2 met15 ura3 fet3::HIS end3::KAN. The parental, End3+ strain constructed was BY4741fet3::HIS3. The pSZ1 plasmid used to assess the presence of the unfolded protein response (UPR) was obtained from Dr. S. Elledge; pSZ1 contains the 22-base pair UPR element that drives expression of lacZ (26Mori K. Sant A. Kohno K. Normington K. Gething M.J. Sambrook J.F. EMBO J. 1992; 11: 2583-2593Crossref PubMed Scopus (310) Google Scholar). Early log phase cells (A660 nm = 0.8-2.0) grown in selective media (6.67 g/liter yeast nitrogen base w/o amino acids, 2% glucose plus the appropriate drop-out mixture of amino acids) were used for all experiments. Plasmid Construction—The parental FET3- and FTR1-containing plasmids (based on pGBT9 and pGAD424, respectively) used for the initial yeast two-hybrid studies were obtained from Professor Takashi Ito (University of Tokyo) (13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The FET3 open reading frame 3′ to the GAL4 DNA binding domain was modified by excising the first 63 nucleotides that encode the ER signal sequence in Fet3p (27Hassett R.F. Yuan D.S. Kosman D.J. J. Biol. Chem. 1998; 273: 23274-23282Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). This vector and the truncation mutants in Gal4 DBD:Fet3p and Gal4 AD:Ftr1p-producing plasmids were created by using a “looping out” approach using the Stratagene QuikChange kit. In this approach a primer was designed so that ∼20 bases annealed immediately 3′ of the region to be excised, and ∼20 bases annealed immediately 5′ of that region. The FET3-containing pRS416 plasmids (28Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) used in this study were derived from pDY133 (11Wang T.-P. Quintanar L. Severance S. Solomon E.I. Kosman D.J. J. Biol. Inorg. Chem. 2003; 8: 611-620Crossref PubMed Scopus (39) Google Scholar). The FTR1-containing plasmids were derived from p703FTR1, a pRS415 vector containing the 2.2-kilobase FTR1 transcription unit (6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar). The FET3:G/C/YFP- and FTR1:G/YFP-containing plasmids were created by cloning the respective fluorescent protein encoding sequences in-frame at the carboxyl termini of full-length FET3 and FTR1 in the plasmids pDY133 and p703FTR1, respectively. The truncation mutants in these plasmids were created by looping out as described earlier. The plasmid used for assessing the FRET between Ctr1p monomers was constructed by amplifying the CTR1 open reading frame from genomic DNA with flanking BglII and XbaI sites that were used to replace the FET3 sequences in the FET3:CFP and FET3:YFP vectors above. The pGAL-Fet5HA plasmid used in this study was a gift of Dr. D. Eide (University of Wisconsin). The FTH1:GFP plasmid was a gift from Dr. R. Piper (University of Iowa). The chimeric plasmids used in this study were created by replacing Fet3p domains with the homologous domains of Fet5p using unique restriction sites created in FET3- and FET5-containing vectors by directed mutagenesis (QuikChange) and standard cloning techniques. Yeast Two-hybrid Analyses—The pGAD-FTR1 and pGBT9-FET3 plasmids and their truncation and point mutant versions were co-transformed in the yeast strain SFY-526, which supports GAL4 promoter-regulated expression of the reporter lacZ gene. The β-galactosidase assays in these transformants were performed as described (29Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). For weak interactions, the yeast two-hybrid plasmid pairs were co-transformed in the yeast strain HF7C, which contains the GAL4 promoter regulating the HIS3 gene. The transformants were replica-plated onto synthetic complete-His-containing plates, and the strength of interaction was determined by replica-plating onto 3-aminotriazole-containing selective plates. Confocal Fluorescent Microscopy—Yeast cells (1 ml) were grown as described, pelleted, washed once with phosphate-buffered saline, and resuspended in 100 μl of the same buffer. Confocal images were obtained using a Bio-Rad MRC 1024 confocal system equipped with a 15-milliwatt krypton/argon laser and operating on a Nikon Optiphot upright microscope with an oil immersion 60× 1.4 NA objective. Optical sections were acquired at 0.5 μm, and xy resolution was set at 0.2 μm using the instrument's Lasersharp Version 3.0 software. Nuclear chromatin and vacuolar membrane double-staining was achieved by incubating cells with DRAQ-5 (50 μm) and FM4-64 (10 μm), respectively. For G/YFP, images were recorded by excitation at 488 nm using a 522/32 emission filter. For FM4-64, excitation was at 568 nm using emission filter 585LP. For DRAQ-5, images were recorded by excitation at 647 nm using a 660LP emission filter. FRET (30Overton M.C. Blumer K.J. Methods. 2002; 27: 324-332Crossref PubMed Scopus (38) Google Scholar)—Starting with a colony from a fresh transformation plate, a culture was grown at 30 ° to an A660 approximately equal to 1.0 and then washed twice with 25 mm Tris, pH 6.8 (FRET buffer). Cells were re-suspended in FRET buffer to give a cell concentration of ∼1.3×108 cells/ml, and 3-ml aliquots were analyzed by scanning fluorometry in a 4.5-ml glass cuvette using a PerkinElmer Life Sciences Model LS50B luminescence spectrometer. For detection of cell autofluorescence, CFP fluorescence, FRET, and YFP fluorescence, an excitation wavelength of 425 nm (λmax for excitation of CFP) was used, and emission was recorded between 450 and 610 nm (slits set at 4.0 nm). For detection of YFP fluorescence at the λmax for excitation of YFP, an excitation wavelength of 510 nm was used, and emission was recorded between 520 and 610 nm (slits set at 4.0 nm). The FL-Win Lab software, Firmware E5, was used to collect the emission spectra, which were the result of signal-averaged values from four scans. Excel spreadsheets and Prism 4.0 by Graph Pad Software (San Diego, CA) were used subsequently to analyze the data as described by Overton and Blumer (30Overton M.C. Blumer K.J. Methods. 2002; 27: 324-332Crossref PubMed Scopus (38) Google Scholar) with minor modifications. Specifically, control cells (no fluorescently tagged proteins expressed) and cells that co-expressed Fet3:CFP and Ftr1:YFP were irradiated at the λmax for excitation of CFP (425 nm). Both spectra were normalized using values obtained at 450-nm emission to adjust for differences in cell concentration. Emission at 450 nm from cells excited at 425 nm is the result of autofluorescence. There is no emission from CFP and YFP at 450 nm. The autofluorescence emission spectrum obtained from control cells was subtracted from the spectrum obtained with cells co-expressing Fet3:CFP and Ftr1: YFP, resulting in a CFP + YFP emission curve (referred to as Curve 1 for convenience). This CFP + YFP emission spectrum is a composite of CFP emission, YFP emission due to direct excitation at 425 nm, and YFP emission due to FRET. Cells expressing only Fet3:CFP were irradiated at the λmax for excitation of CFP. This spectrum is normalized at 476 nm to give a CFP emission peak value identical to that of Curve 1. After normalization, the normalized CFP spectrum is subtracted from Curve 1. Subtracting the CFP curve results in a YFP emission spectrum (Curve 2) composed of a FRET component and another component due to direct excitation of YFP. The YFP component of Curve 2 that is due to the direct excitation of YFP at 425 nm must be subtracted from Curve 2 to obtain the YFP emission spectrum due only to FRET (the indirect excitation of YFP at 425 nm). The YFP emission spectra obtained from cells producing only Ftr1:YFP irradiated at 425 nm is subtracted from Curve 2. The YFP emission spectra of cells co-expressing Fet3:CFP and Ftr1:YFP versus cells expressing only Ftr1:YFP were normalized for differences in YFP expression level by irradiating these two types of cells at the λmax for YFP (510 nm) and recording their respective YFP emission spectra. Because CFP is not excited at this wavelength, these emission spectra quantify the level of only Ftr1:YFP. The ratio of the YFP emission peak heights of these two spectra was used to normalize the YFP emission spectrum due specifically to FRET. The product of this final multiplication step is the emission envelope due specifically to YFP excitation by CFP. Two spectra obtained during the process of generating the FRET emission spectrum are used to calculate the apparent efficiency of FRET; they are the YFP emission spectrum due specifically to FRET and the YFP emission spectrum obtained by irradiating cells co-expressing CFP- and YFP-tagged proteins at the λmax for YFP, 510 nm. Apparent FRET efficiency is calculated by dividing the integrated area of the FRET spectrum by the integrated area of the YFP emission spectrum obtained by excitation at the λmax for YFP. This efficiency is apparent because in our analysis we did not take into account the quenching of CFP fluorescence by YFP (26Mori K. Sant A. Kohno K. Normington K. Gething M.J. Sambrook J.F. EMBO J. 1992; 11: 2583-2593Crossref PubMed Scopus (310) Google Scholar). Oxidase and 59Fe Uptake Assays—For oxidase assays (18Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (393) Google Scholar), cells grown as described were collected by centrifugation and washed once with phosphate-buffered saline. The pellet was resuspended in 200 μl of extraction buffer (150 mm NaCl, 25 mm Tris-HCL, pH 7.4, containing 4 μl of fungal protease inhibitor mix from Sigma). To the mix 200 μl of acid-washed glass beads were added and vortexed for 4 min at 4 °. After removal of large particles, homogenates were centrifuged at 16,000 × g at 4 ° for 30 min. Pellets were washed in extraction buffer, resuspended in buffer containing 1% Triton X-100, and kept on ice for 12 h. The mix was centrifuged at 16,000 × g at 4 ° for 30 min to yield a clarified extract. Extracts were stored at -20 °. Membrane protein extract (30 μg of total protein, determined by Bio-Rad (Bradford) protein assay) in the absence or presence of CuSO4 (50 μm) was mixed with Laemmli sample buffer lacking dithiothreitol. The samples were applied to a 10% SDS/PAGE gel without prior heating and electrophoresed for 3 h at 80 V. The gel was then equilibrated with 50 volumes of 0.05% Triton X-100 in 10% (w/v) glycerol and soaked for an equal time in 5 volumes of 3 mm p-phenylenediamine dihydrochloride (Sigma) in 100 mm sodium acetate, pH 5.7. The gel was then placed in a humid chamber in the dark for at least 24 h and photographed. 59Fe uptake was quantified at [59Fe] = 0.2 μm (≈KmFe) at pH 6.0 in the presence of 20 mm ascorbate (reductase-independent uptake) as described (8Severance S. Chakraborty S. Kosman D.J. Biochem. J. 2004; 380: 487-496Crossref PubMed Google Scholar). Identification of Fet3p·Ftr1p Interaction Motifs—An in vivo interaction between Fet5p and Fth1p in the yeast vacuolar membrane has been documented by co-immunoprecipitation assay (13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This pair of proteins was used as a positive control in a yeast two-hybrid analysis of the interaction between Fet3p and Ftr1p. In this assay the Gal4 DBD-containing vector (the bait) produced a carboxyl-terminal fusion to either Fet3 or Fet5 proteins that lacked their respective putative amino-terminal ER targeting signal sequence but were otherwise full-length. The Gal4 AD-containing vector (the catch) produced a carboxyl-terminal fusion to full-length Ftr1p or Fth1p. Consistent with the co-immunoprecipitation (13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and affinity trap results (12Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3998) Google Scholar), the Fet5p·Fth1p pair did support His+growth in the presence of up to 3 mm 3-aminotriazole and was scored as β-Gal+in the standard strain background used. The Fet3p·Ftr1p pair supported essentially the same qualitative res" @default.
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• W1966642383 title "Assembly, Activation, and Trafficking of the Fet3p·Ftr1p High Affinity Iron Permease Complex in Saccharomyces cerevisiae" @default.
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• W1966642383 doi "https://doi.org/10.1074/jbc.m512042200" @default.
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