The Monocarboxylate Transporter Homolog Mch5p Catalyzes Riboflavin (Vitamin B2) Uptake in Saccharomyces cerevisiae
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m505002200
ISSN1083-351X
Autores Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoRiboflavin is a water-soluble vitamin (vitamin B2) required for the production of the flavin cofactors FMN and FAD. Mammals are unable to synthesize riboflavin and need a dietary supply of the vitamin. Riboflavin transport proteins operating in the plasma membrane thus have an important role in the absorption of the vitamin. However, their sequences remained elusive, and not a single eukaryotic riboflavin transporter is known to date. Here we used a genetic approach to isolate MCH5, a Saccharomyces cerevisiae gene with homology to mammalian monocarboxylate transporters, and characterize the protein as a plasma membrane transporter for riboflavin. This conclusion is based on the suppression of riboflavin biosynthetic mutants (rib mutants) by overexpression of MCH5 and by synthetic growth defects caused by deletion of MCH5 in rib mutants. We also show that cellular processes in multiple compartments are affected by deletion of MCH5 and localize the protein to the plasma membrane. Transport experiments in S. cerevisiae and Schizosaccharomyces pombe cells demonstrate that Mch5p is a high affinity transporter (Km = 17 μm) with a pH optimum at pH 7.5. Riboflavin uptake is not inhibited by protonophores, does not require metabolic energy, and operates by a facilitated diffusion mechanism. The expression of MCH5 is regulated by the cellular riboflavin content. This indicates that S. cerevisiae has a mechanism to sense riboflavin and avert riboflavin deficiency by increasing the expression of the plasma membrane transporter MCH5. Moreover, the other members of the MCH gene family appear to have unrelated functions. Riboflavin is a water-soluble vitamin (vitamin B2) required for the production of the flavin cofactors FMN and FAD. Mammals are unable to synthesize riboflavin and need a dietary supply of the vitamin. Riboflavin transport proteins operating in the plasma membrane thus have an important role in the absorption of the vitamin. However, their sequences remained elusive, and not a single eukaryotic riboflavin transporter is known to date. Here we used a genetic approach to isolate MCH5, a Saccharomyces cerevisiae gene with homology to mammalian monocarboxylate transporters, and characterize the protein as a plasma membrane transporter for riboflavin. This conclusion is based on the suppression of riboflavin biosynthetic mutants (rib mutants) by overexpression of MCH5 and by synthetic growth defects caused by deletion of MCH5 in rib mutants. We also show that cellular processes in multiple compartments are affected by deletion of MCH5 and localize the protein to the plasma membrane. Transport experiments in S. cerevisiae and Schizosaccharomyces pombe cells demonstrate that Mch5p is a high affinity transporter (Km = 17 μm) with a pH optimum at pH 7.5. Riboflavin uptake is not inhibited by protonophores, does not require metabolic energy, and operates by a facilitated diffusion mechanism. The expression of MCH5 is regulated by the cellular riboflavin content. This indicates that S. cerevisiae has a mechanism to sense riboflavin and avert riboflavin deficiency by increasing the expression of the plasma membrane transporter MCH5. Moreover, the other members of the MCH gene family appear to have unrelated functions. The proteins that require FMN or FAD as cofactors are termed flavoproteins. Mostly, they contain noncovalently bound flavin cofactors and are specific for either FAD or FMN. Some of them contain auxiliary groups such as pteridin, heme, iron-sulfur centers, molybdenum, or other metal ions or contain disulfides in their active site. Flavoproteins are involved in a wide range of biochemical reactions. They play a pivotal role in the dehydrogenation of metabolites in one- and two-electron transfer reactions from and to redox centers, in the activation of oxygen for oxidation, and in hydroxylation reactions (1Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). The flavin cofactor FMN is produced from riboflavin by the action of riboflavin kinase. FAD derives from FMN and ATP, a reaction catalyzed by FAD synthetase. Riboflavin is a vitamin (vitamin B2) for mammals and many other organisms. Thus, dietary riboflavin has to be taken up from the gut and then provided to every single cell in a multicellular organism. Plasma membrane riboflavin transporters are thought to play an important role in the distribution of riboflavin. However, their existence in many cell types up to now has only been demonstrated biochemically (reviewed in Ref. 2Foraker A.B. Khantwal C.M. Swaan P.W. Adv. Drug Delivery Rev. 2003; 55: 1467-1483Crossref PubMed Scopus (102) Google Scholar). Whereas passive uptake of riboflavin is commonly observed in riboflavin-sufficient conditions, riboflavin uptake at low concentrations follows saturation kinetics and displays high affinity for the substrate (Km = 1nm to 1 μm (2Foraker A.B. Khantwal C.M. Swaan P.W. Adv. Drug Delivery Rev. 2003; 55: 1467-1483Crossref PubMed Scopus (102) Google Scholar)). Riboflavin is not required by most fungal organisms (3Koser S.A. Vitamin Requirements of Bacteria and Yeasts. Charles C. Thomas, Springfield, IL1968Google Scholar), indicating that most of them produce riboflavin. Indeed, the yeast Saccharomyces cerevisiae is known to be an excellent dietary source of riboflavin (4Bässler K.-H. Golly I. Loew D. Pietrzuk K. Vitamin-Lexikon. 3rd Ed. Urban & Fischer, München, Germany2002Google Scholar) and to possess all enzymes required for riboflavin biosynthesis, which are encoded by the RIB genes (RIB1, RIB2, RIB3, RIB4, RIB5, and RIB7). Some fungal organisms are utilized in commercial riboflavin production processes. One of the best riboflavin producers is the filamentous hemiascomycete Ashbya gossypii that is capable of producing the astonishing amount of 15 g of riboflavin/liter of medium, a concentration at which riboflavin readily crystallizes (5Stahmann K.P. Revuelta J.L. Seulberger H. Appl. Microbiol. Biotechnol. 2000; 53: 509-516Crossref PubMed Scopus (290) Google Scholar). Other riboflavin overproducers include mutants of the yeasts Candida famata, Pichia guilliermondii, or engineered Bacillus subtilis strains (5Stahmann K.P. Revuelta J.L. Seulberger H. Appl. Microbiol. Biotechnol. 2000; 53: 509-516Crossref PubMed Scopus (290) Google Scholar, 6Sibirny A.A. Nonconventional Yeasts in Biotechnology.in: Wolf K. Springer, Berlin, Germany1996: 255-275Crossref Google Scholar). Despite of being able to synthesize riboflavin, fungal organisms are also able to take up riboflavin from the culture medium. This activity was recently analyzed in an Ashbya gossypii rib5Δ mutant defective in the last step of riboflavin biosynthesis (7Förster C. Revuelta J.L. Kramer R. Appl. Microbiol. Biotechnol. 2001; 55: 85-89Crossref PubMed Scopus (23) Google Scholar). Riboflavin uptake was found to be a high affinity (Km = 40 μm) process with low overall activity. The activity was sensitive to competition by FAD and FMN and to inhibition by 2,4-dinitrophenol, an uncoupler of the transmembrane proton gradient (7Förster C. Revuelta J.L. Kramer R. Appl. Microbiol. Biotechnol. 2001; 55: 85-89Crossref PubMed Scopus (23) Google Scholar). Riboflavin auxotrophic S. cerevisiae mutants were also found to take up extracellular riboflavin with high affinity (Km = 15 μm) and specificity, but no energy requirement was apparent (8Perl M. Kearney E.B. Singer T.P. J. Biol. Chem. 1976; 251: 3221-3228Abstract Full Text PDF PubMed Google Scholar). Riboflavin uptake has also been shown in riboflavin requiring mutants of the yeast Pichia guilliermondii (9Sibirnyi A.A. Shavlovskii G.M. Ksheminskaia G.P. Orlovskaia A.G. Biokhimiia. 1977; 42: 1841-1851PubMed Google Scholar). Despite this biochemical evidence, not a single eukaryotic riboflavin transporter is known to date. Here, we used a classical genetic approach and identified Mch5p, a yeast protein with homology to mammalian monocarboxylate transporters (MCTs), 3The abbreviations used are: MCTmonocarboxylate transporterORFopen reading frameCPYcarboxypeptidase Y. as the plasma membrane transporter for riboflavin. monocarboxylate transporter open reading frame carboxypeptidase Y. Yeast Strains—Yeast strains were derived from BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (10Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar)). Deletion mutants were obtained from EUROSCARF (Frankfurt/Main, Germany). Haploid rib2Δ, rib3Δ, rib5Δ, and rib7Δ strains were generated by sporulation of heterozygous diploids, followed by tetrad dissection on media containing 200 mg/liter riboflavin. Double mutants were generated by PCR amplification of the rib4Δ::kanMX4 or rib5Δ::kanMX4 alleles from deletion mutants. After ligation of the PCR products into a pUC19 derivative, kanMX4 was exchanged for the Schizosaccharomyces pombe his5+ gene from plasmid pFA6a-HIS3MX6 (11Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). The constructs were excised and used to transform S. cerevisiae cells to histidine prototrophy. A deletion construct for MCH5 was made by ligating a SalI-EcoRI fragment from a library plasmid into pBluescript. The plasmid was cut with BglII, and URA3 was inserted as a BamHI fragment from YDp-U (12Berben G. Dumont J. Gilliquet V. Bolle P.A. Hilger F. Yeast. 1991; 7: 475-477Crossref PubMed Scopus (317) Google Scholar). Next, the mch5Δ::URA3 construct was excised with SalI and EcoRI and used for transformation. In every case, correct deletion was confirmed by PCR. CEN.PK113–13D (MATa ura3–52 MAL2–8c SUC2) and the mch1-mch5Δ deletion mutant in the CEN.PK background (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar) were a kind gift from Eckhard Boles (Universität Frankfurt/Main, Germany). The rib5Δ::kanMX4 mutant in CEN.PK was made by transformation of a PCR product generated from rib5Δ::kanMX in the BY strain background. The mch5Δ::URA3 mutant was generated as outlined above. The S. pombe strain used for the expression of MCH5 was FY254 (ade6-M210 can1-1 leu1-32 ura4-D18 h– (14Liang D.T. Hodson J.A. Forsburg S.L. J. Cell Sci. 1999; 112: 559-567Crossref PubMed Google Scholar)). Media and Growth Conditions—Yeast media were YPD (2% peptone, 2% glucose, 1% yeast extract), SD-Glc (2% glucose, 0.67% yeast nitrogen base without amino acids (YNB) or SD-Gal (2% galactose, 0.67% YNB). SD media contain 200 μg/liter riboflavin and were supplemented with additional riboflavin to give the desired final concentration. Riboflavin-free media were made from yeast nitrogen base without amino acids without vitamins (Bio 101) and contained all vitamins except riboflavin in standard concentrations. As a rule, only supplements that were required by the strains were added. YADE (2% yeast extract, 0.2% ammonium sulfate, 0.2% glucose, and 3% ethanol) was used for the preparation of mitochondria. S. pombe cells were grown in EMM, a standard synthetic medium for S. pombe, which is devoid of riboflavin (15Mitchison J.M. Methods Cell Physiol. 1970; 4: 131-165Crossref Scopus (286) Google Scholar). Suspensions of yeast cells for use in growth assays were prepared in 96-well plates. Serial 10-fold dilutions starting from A600 = 0.6 in adjoining wells were transferred to agar plates using a stainless steel replication device. Growth was recorded after incubation at 30 °C. Plasmids—An S. cerevisiae library in the multicopy vector YEp352 was used (16te Heesen S. Knauer R. Lehle L. Aebi M. EMBO J. 1993; 12: 279-284Crossref PubMed Scopus (106) Google Scholar). As illustrated in Fig. 1A, YOR305W was isolated from a complementing library plasmid as BglII/SalI (polylinker site) fragment and ligated into the BamHI/SalI sites of the URA3 2μ plasmid pRS426 (17Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1438) Google Scholar). To isolate YOR306C (MCH5), we used a SalI/EcoRI restriction fragment, which was ligated into the same sites of pRS426. For transport assays in S. cerevisiae, MCH5 was amplified from a complementing library plasmid and cloned downstream of the TDH3 promotor in plasmid p426-TDH (18Mumberg D. Müller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar). N-terminal tagging of MCH5 was performed in the low copy plasmid pRS316 by fusing a GAL1 promotor to a 9-Myc tag, followed by the MCH5 ORF lacking the ATG, followed by 224 bp of MCH5 downstream sequence. As assayed by growth of rib4Δ and rib5Δ mutants on low concentrations of riboflavin, both constructs encoded functional versions of Mch5p (data not shown). Reporter assays were performed with cells transformed with a construct containing 832 bp of MCH5 5′-sequences (obtained as a PstI/SphI cut PCR product), which were fused to the E. coli lacZ gene (a SphI/XbaI cut PCR product generated from genomic DNA) in the URA3/CEN plasmid YCplac33 (19Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar). To express MCH1, MCH2, MCH3, MCH4, or MCH5 at similar levels in S. cerevisiae, we amplified each ORF from genomic DNA with primers that added restriction sites adjacent to the start and to the stop codon. The PCR products were ligated into pPCR-Skript Amp (Stratagene), sequence-verified, and finally cloned downstream of the galactose-inducible GAL1 promotor of the multicopy vector pYES2 (Invitrogen). S. pombe expression used the thiamin-repressible pREP3X vector (20Forsburg S.L. Nucleic Acids Res. 1993; 21: 2955-2956Crossref PubMed Scopus (401) Google Scholar), into which MCH5 was cloned as a PCR product. Uptake Experiments—S. cerevisiae rib4Δ for use in uptake experiments were grown overnight in SD media containing 20 mg/liter riboflavin. The cells were washed, suspended in SD lacking riboflavin for A600 = 0.2, and shaken for at least 8 h at 30 °C. S. pombe cells were grown to midlogarithmic phase in EMM. All cells were washed with water, resuspended in 40 mm K2HPO4/KH2PO4, 10 mm KCl, pH 7.5, and stored on ice. Uptake experiments were performed at 30 °C in a volume of 500 μl containing five OD cells, 40 mm K2HPO4/KH2PO4, pH 7.5, 10 mm KCl, and 3.2 μm [14C]riboflavin (specific activity 5.54 MBq/mg; a gift of Prof. Dr. Reinhard Krämer, Universität zu Köln, Germany). Aliquots were removed at timed intervals, filtered on glass fiber filters, washed with cold water, and analyzed by scintillation counting. We observed a distinct day-to-day variability in overall riboflavin transport activity in both expression systems, which restricted us to only compare results obtained with the same batch of cells. Additional controls ensured that storage on ice preserved the transport activity of the cells. Glucose uptake was performed as in Ref. 21Bisson L.F. Fraenkel D.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1730-1734Crossref PubMed Scopus (217) Google Scholar, biotin uptake as in Ref. 22Stolz J. Hoja U. Meier S. Sauer N. Schweizer E. J. Biol. Chem. 1999; 274: 18741-18746Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar. Other Experimental Procedures—The following procedures were performed according to published protocols: pulse-chase labeling of carboxypeptidase Y (CPY) (23Tu B.P. Ho-Schleyer S.C. Travers K.J. Weissman J.S. Science. 2000; 290: 1571-1574Crossref PubMed Scopus (355) Google Scholar), cell fractionation (24Opekarova M. Robl I. Tanner W. Biochim. Biophys. Acta. 2002; 1564: 9-13Crossref PubMed Scopus (36) Google Scholar), lacZ assays using permeabilized cells (25Rupp S. Methods Enzymol. 2002; 350: 112-131Crossref PubMed Scopus (41) Google Scholar), and succinate dehydrogenase assays (26Valle A.B. Panek A.D. Mattoon J.R. Anal. Biochem. 1978; 91: 583-599Crossref PubMed Scopus (11) Google Scholar). MCH5 Shows Genetic Interactions with Riboflavin Biosynthetic Genes—To isolate the S. cerevisiae plasma membrane riboflavin transporter, we first analyzed the growth phenotype of mutants with defects in riboflavin biosynthesis. The rib4Δ and rib5Δ mutants are defective in the penultimate and last step of riboflavin biosynthesis, respectively. When compared with wild-type cells, both mutants showed growth defects on plates containing low concentrations of riboflavin (Fig. 1B). Surprisingly, for two enzymes catalyzing consecutive steps in the same biochemical pathway, rib4Δ and rib5Δ cells behaved differently. On minimal plates, rib4Δ cells displayed a milder phenotype and could grow with 2 mg/liter riboflavin. rib5Δ cells, in contrast, required a 10-fold higher concentration of riboflavin for growth (Fig. 1B). Similarly, rib4Δ could grow on complete media (YPD) without added riboflavin, whereas only a supplement of 20 mg/liter of riboflavin allowed growth of rib5Δ (Fig. 1C). The milder phenotype of rib4Δ is probably explained by the fact that the reaction catalyzed by Rib4p proceeds without enzymatic catalysis (27Kis K. Kugelbrey K. Bacher A. J. Org. Chem. 2001; 66: 2555-2559Crossref PubMed Scopus (34) Google Scholar). Thus, although both mutants showed a different behavior, both had strong growth defects on low riboflavin plates. Importantly, both appeared to grow normally when their riboflavin requirement was met (Fig. 1, B and C). The strong growth defects of rib4Δ and rib5Δ mutants initiated a genetic screen in which the riboflavin transporter was searched as a multicopy suppressor of the rib mutants. To this end, both mutants were transformed with a multicopy genomic library, followed by plating on media containing 0.2 (rib4Δ) or 2 mg/liter (rib5Δ) riboflavin. Plasmid DNA was isolated from colonies that were able to grow on these media and retransformed into rib4Δ or rib5Δ mutants. Plasmids that allowed growth on plates containing no riboflavin were eliminated because they probably contained the riboflavin biosynthesis genes defective in the mutants. For random samples, this was confirmed by restriction analysis or PCR. The remaining plasmids, 4 of 20,000 tranformants in rib4Δ and 10 of 36,000 transformants in rib5Δ, did not allow growth on riboflavin-free plates and showed partial correction of the growth defects of the mutants. All complementing plasmids contained fragments from the same region of chromosome XV, and the smallest plasmid harbored only two ORFs, YOR305W and YOR306C (Fig. 1A). Only little information is available on YOR305W. The ORF appears to be present in other budding yeast species and cause slow growth when deleted, making it unlikely that it is a spurious ORF (28Christie K.R. Weng S. Balakrishnan R. Costanzo M.C. Dolinski K. Dwight S.S. Engel S.R. Feierbach B. Fisk D.G. Hirschman J.E. Hong E.L. Issel-Tarver L. Nash R. Sethuraman A. Starr B. Theesfeld C.L. Andrada R. Binkley G. Dong Q. Lane C. Schroeder M. Botstein D. Cherry J.M. Nucleic Acids Res. 2004; 32: D311-D314Crossref PubMed Google Scholar). YOR306C, in contrast, was previously characterized to be a yeast homolog of mammalian monocarboxylate transporters (MCTs; reviewed in Ref. 29Halestrap A.P. Price N.T. Biochem. J. 1999; 343: 281-299Crossref PubMed Scopus (1100) Google Scholar) and hence named MCH5 (for monocarboxylate transporter homolog 5 (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar)). To analyze which of the two ORFs were responsible for the suppressor activity, we individually subcloned MCH5 and YOR305W from the library plasmid into the multicopy plasmid pRS426 (Fig. 1A). We transformed these plasmids into rib4Δ and rib5Δ mutants and analyzed the riboflavin requirement of the transformed cells (Fig. 1B). Of the two genes present on the library plasmid, only MCH5 was able to suppress the growth phenotype resulting from loss of RIB4 or RIB5 (Fig. 1B). Expression of MCH5, however, did not fully restore growth of rib4Δ and rib5Δ back to wild-type levels. Whereas wild-type growth rates were typical for strains that contained the authentic RIB genes on library plasmids, overexpression of MCH5 did not allow growth in the absence of riboflavin and allowed somewhat slower growth on plates containing 0.2 mg/liter riboflavin (Fig. 1B and data not shown). We also transformed the MCH5 containing plasmid into rib2Δ, rib3Δ, and rib7Δ deletion mutants. Before transformation, these strains behaved like rib5Δ and required 20 mg/liter riboflavin for growth. After transformation with pRS426-MCH5, all of them were able to grow on plates containing 2 mg/liter riboflavin (data not shown). In conclusion, our experiments establish that the suppressor activity identified in the screening is carried by MCH5 and not by YOR305W. Moreover, MCH5 was able to suppress all rib mutants when overexpressed. To analyze the interaction of MCH5 with riboflavin biosynthesis genes in greater detail, we generated mch5Δ rib4Δ and mch5Δ rib5Δ double mutants and analyzed their riboflavin requirements (Fig. 1C). Deletion of MCH5 in a wild-type strain did not lead to a phenotype. In contrast, deletion of MCH5 increased the riboflavin requirements of rib4Δ and rib5Δ (Fig. 1C). Surprisingly, mch5Δ rib4Δ and mch5Δ rib5Δ strains showed identical growth on YPD plates containing 20 mg/liter riboflavin (Fig. 1C), whereas of the corresponding single rib mutants, rib4Δ showed much better growth. Although the reason for this is not known, this may indicate that the mch5Δ rib4Δ double mutant can perform the chemical reaction that bypasses Rib4p only with reduced efficiency. In conclusion, overexpression of MCH5 rescues the growth defects of rib mutants, whereas their phenotype is exacerbated by deletion of MCH5. The Function of MCH5 Is Unique within the MCH Gene Family—Mch5p is a 521-amino acid protein with 12 hydrophobic regions probably corresponding to 12 transmembrane domains (30Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar). It is a member of a yeast protein family consisting of five members (Mch1p–Mch5p (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar)) and is most similar to Mch4p with which it shares 45% identical amino acids (31Nelissen B. De Wachter R. Goffeau A. FEMS Microbiol. Rev. 1997; 21: 113-134Crossref PubMed Google Scholar). Of the MCH genes, MCH5 appears to have the highest expression level, followed by MCH4 (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar). To analyze if the function of MCH5 was shared by any of the other MCH genes, we individually expressed the MCH1, MCH2, MCH3, and MCH4 ORFs in rib5Δ. To achieve similar levels of expression, all ORFs were amplified from genomic DNA and cloned downstream of the GAL1 promotor in the multicopy plasmid pYES2. Analysis of the transformants revealed that only MCH5 allowed growth of rib5Δ cells on low riboflavin concentrations (Fig. 1D). Moreover, suppression by MCH5 was not seen on glucose-containing plates, indicating that expression of MCH5 was necessary for its activity as a multicopy suppressor (Fig. 1D). We also deleted RIB5 in individual strains carrying null alleles of MCH1, MCH2, MCH3, MCH4, or MCH5 and found synthetic defects only for the combined deletion of MCH5 and RIB5 (data not shown). These findings were corroborated by growth assays performed with a strain deleted for all five MCH genes (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar). Because this strain was from the CEN.PK background, we generated a series of isogenic strains deleted for RIB5, RIB5 and MCH5, or RIB5 and all MCH genes and used them in growth assays (Fig. 1E). We found that mch1–5Δ showed wild-type growth, but growth was affected by deletion of RIB5. As expected from our findings in the BY strain background (Fig. 1C), deletion of MCH5 further reduced the growth of rib5Δ. Importantly, a strain deleted for all MCH genes and for RIB5 showed no growth defects beyond the defects seen in mch5Δ rib5Δ (Fig. 1E). This demonstrates that MCH5 performs a unique function within the yeast MCH gene family. This function, however, appears to be different from the human monocarboxylate transporters. Whereas the human proteins act as transporters for pyruvate, lactate, ketone bodies, and other monocarboxylates (29Halestrap A.P. Price N.T. Biochem. J. 1999; 343: 281-299Crossref PubMed Scopus (1100) Google Scholar), these substances were ruled out as possible substrates of the yeast Mch proteins (13Makuc J. Paiva S. Schauen M. Kramer R. Andre B. Casal M. Leao C. Boles E. Yeast. 2001; 18: 1131-1143Crossref PubMed Scopus (56) Google Scholar). ribΔ Strains Develop Extragenic Suppressor Mutations—In the course of our experiments, we noted that strains with ribΔ mutations frequently segregated colonies that display improved growth (see Figs. 1, C and D). This behavior has been observed by others for rib4Δ and was interpreted to be due to the appearance of extragenic supressors (32Lesuisse E. Knight S.A. Courel M. Santos R. Camadro J.M. Dancis A. Genetics. 2005; 169: 107-122Crossref PubMed Scopus (43) Google Scholar). Initially, we identified suppressor mutants only in rib4Δ, but this finding was likely influenced by the higher riboflavin content of rib4Δ, relative to the other ribΔ mutants. Closer investigation of this phenomenon provided evidence that colonies with improved growth characteristics arise at similar frequencies in all riboflavin biosynthetic mutants tested. 4A. Spitzner, P. Reihl, and J. Stolz, unpublished observations. Whereas this high frequency of spontaneous mutations in ribΔ mutants is probably explained by the involvement of the riboflavin biosynthetic pathway in the detoxification of 8-oxo-GTP, a spontaneously produced mutagenic substrate for DNA synthesis (33Kobayashi M. Ohara-Nemoto Y. Kaneko M. Hayakawa H. Sekiguchi M. Yamamoto K. J. Biol. Chem. 1998; 273: 26394-26399Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 34Boretsky Y.R. Kapustyak K.Y. Fayura L.R. Stasyk O.V. Stenchuk M.M. Bobak Y.P. Drobot L.B. Sibirny A.A. FEMS Yeast Res. 2005; 5: 829-837Crossref PubMed Scopus (20) Google Scholar), the precise mechanism by which the suppressor mutations improve growth is currently unknown. Suppressor mutations also arise in ribΔ mch5Δ strains, indicating that suppression is not caused by a mutation in MCH5 (Fig. 1C). Due to this phenomenon, the strains used for the analyses described below were taken from plates that were free of suppressors and their growth in liquid media was limited to the minimal time required to perform the experiment. Moreover, we carefully monitored the growth rates to exclude cultures that grew faster then expected from further analysis. A detailed analysis of the suppressor mutants will be presented elsewhere. Deletion of MCH5 Causes Inactivation of FAD-dependent Processes—To assess the consequences caused by inactivation of RIB5 and MCH5, we determined the activity of FAD dependent processes located in different cellular compartments. It has been demonstrated before that the formation of disulfide bonds in proteins depends on riboflavin biosynthesis (23Tu B.P. Ho-Schleyer S.C. Travers K.J. Weissman J.S. Science. 2000; 290: 1571-1574Crossref PubMed Scopus (355) Google Scholar) and is catalyzed by the FAD-dependent oxidase Ero1p localized in the ER lumen (35Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 36Tu B.P. Weissman J.S. Mol. Cell. 2002; 10: 983-994Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). A reduced capability to oxidize protein thiols results in the ER retention of disulfide-containing proteins after treatment of cells with reducing agents such as dithiothreitol (23Tu B.P. Ho-Schleyer S.C. Travers K.J. Weissman J.S. Science. 2000; 290: 1571-1574Crossref PubMed Scopus (355) Google Scholar). CPY, a vacuolar protease containing five disulfides, is a convenient marker to monitor protein oxidation, because disulfide formation is required for the folding and ER export and because its electrophoretic mobility reflects its cellular localization (23Tu B.P. Ho-Schleyer S.C. Travers K.J. Weissman J.S. Science. 2000; 290: 1571-1574Crossref PubMed Scopus (355) Google Scholar, 35Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 37Stevens T. Esmon B. Schekman R. Cell. 1982; 30: 439-448Abstract Full Text PDF PubMed Scopus (371) Google Scholar). Pulse-chase experiments revealed that wild-type cells matured CPY very quickly (Fig. 2A). Although the CPY precursors (p1, corresponding to the ER form, and p2, corresponding to the Golgi form) were more abundant, the vacuolar (m) form of CPY could already be detected 10 min after dithiothreitol removal. Precursors were only present in trace amounts at later time points, where the mature form accumulated as expected (Fig. 2A). rib5Δ mutants showed a slower maturation of CPY. After a 10-min chase, no mature CPY was present and only p1 and p2 forms were observed. In rib5Δ, precursor and mature forms showed roughly equal abundance after a 30-min chase, whereas at 60 min and later, mCPY was the dominant form, and precursors had a lower abundance. Maturation of CPY was further slowed in rib5Δ mch5Δ double mutants where mature forms appeared only after a 60-min chase. It was also evident from Fig. 2A that rib5Δ mch5Δ do not produce the p2 form of CPY. This can be explained by assuming that only CP
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