RESUMEN
Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected.NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.
Asunto(s)
Dipéptidos/metabolismo , Endopeptidasa K/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Simportadores/metabolismo , Animales , Transporte Biológico , Biotinilación , Línea Celular , Glicosilación , Cinética , Potenciales de la Membrana , Ratones , Mutación , Transportador de Péptidos 1 , Estabilidad Proteica , Simportadores/genética , Transfección , Tripsina/metabolismo , Xenopus laevisRESUMEN
Although increased dietary fructose consumption is associated with metabolic impairments, the mechanisms and regulation of intestinal fructose absorption are poorly understood. GLUT5 is considered to be the main intestinal fructose transporter. Other GLUT family members, such as GLUT7 and GLUT9 are also expressed in the intestine and were shown to transport fructose and glucose. A conserved isoleucine-containing motif (NXI) was proposed to be essential for fructose transport capacity of GLUT7 and GLUT9 but also of GLUT2 and GLUT5. In assessing whether human GLUT2, GLUT5, GLUT7, and GLUT9 are indeed fructose transporters, we expressed these proteins in Xenopus laevis oocytes. Stably transfected NIH-3T3 fibroblasts were used as second expression system. In proving the role of the NXI motif, variants p.I322V of GLUT2 and p.I296V of GLUT5 were tested as well. Sugar transport was measured by radiotracer flux assays or by metabolomics analysis of cell extracts by GC-MS. Fructose and glucose uptakes by GLUT7 were not increased in both expression systems. In search for the physiological substrate of GLUT7, cells overexpressing the protein were exposed to various metabolite mixtures, but we failed to identify a substrate. Although urate transport by GLUT9 could be shown, neither fructose nor glucose transport was detectable. Fructose uptake was decreased by the GLUT2 p.I322V variant, but remained unaffected in the p.I296V GLUT5 variant. Thus, our work does not find evidence that GLUT7 or GLUT9 transport fructose or glucose or that the isoleucine residue determines fructose specificity. Rather, the physiological substrate of GLUT7 awaits to be discovered.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Animales , Transporte Biológico/genética , Transporte Biológico/fisiología , Fructosa/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Ratones , Microscopía Fluorescente , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Células 3T3 NIH , Oocitos/metabolismo , Xenopus laevisRESUMEN
Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.
Asunto(s)
Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Nucleobases/metabolismo , Piridoxina/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Expresión Génica , Prueba de Complementación Genética , Microscopía Confocal , Familia de Multigenes , Mutación , Proteínas de Transporte de Nucleobases/genética , Fenotipo , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Epidermis de la Planta/ultraestructura , Exudados de Plantas/análisis , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantas Modificadas Genéticamente , Piridoxina/química , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: The bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain. RESULTS: The gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-ß-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG. CONCLUSIONS: The energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain (or any other microorganism).
Asunto(s)
Bacillus subtilis , Reactores Biológicos , Biotecnología/métodos , Proteínas de Transporte de Membrana/metabolismo , Riboflavina/biosíntesis , Streptomyces/metabolismo , Northern Blotting , Western Blotting , Radioisótopos de Carbono , Escherichia coli , Técnicas de Transferencia de Gen , Isopropil Tiogalactósido , Proteínas de Transporte de Membrana/genética , Plásmidos/genética , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Streptomyces/genéticaRESUMEN
In Salmonella enterica serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for myo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of iolT1 (STM4418) led to a severe growth defect, and deletion of iolT1 (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes in trans. Bioluminescence-based reporter assays demonstrated a strong induction of their promoters P(iolT1) and P(iolT2) in the presence of MI but not of glucose. Deletion of iolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of P(iolT1) and P(iolT2), indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in Escherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using myo-[1,2-[(3)H](N)]inositol, a saturable uptake activity of IolT1 with a K(m) value between 0.49 and 0.79 mM was determined in DH5alpha expressing IolT1, in S. enterica serovar Typhimurium strain 14028, and in mutant 14028 DeltaiolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI.
Asunto(s)
Proteínas Bacterianas/metabolismo , Islas Genómicas , Inositol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Salmonella typhimurium/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B(1)). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDDeltathiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.
Asunto(s)
Proteínas Bacterianas/metabolismo , Listeria monocytogenes/crecimiento & desarrollo , Listeriosis/microbiología , Proteínas de Transporte de Membrana/metabolismo , Tiamina/biosíntesis , Proteínas Bacterianas/genética , Transporte Biológico , Células CACO-2 , Células Epiteliales/microbiología , Humanos , Iminofuranosas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Proteínas de Transporte de Membrana/genética , Unión ProteicaRESUMEN
Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B2). Riboflavin auxotrophic mutants with deletions in any of the RIB genes frequently segregate colonies with improved growth. We demonstrate by reporter assays and Western blots that these suppressor mutants overexpress the plasma-membrane riboflavin transporter MCH5. Frequently, this overexpression is mediated by the transcription factor Put3, which also regulates the proline catabolic genes PUT1 and PUT2. The increased expression of MCH5 may increase the concentrations of FAD, which is the coenzyme required for the activity of proline oxidase, encoded by PUT1. Thus, Put3 regulates proline oxidase activity by synchronizing the biosynthesis of the apoenzyme and the coenzyme FAD. Put3 is known to bind to the promoters of PUT1 and PUT2 constitutively, and we demonstrate by gel-shift assays that it also binds to the promoter of MCH5. Put3-mediated transcriptional activation requires proline as an inducer. We find that the increased activity of Put3 in one of the suppressor mutants is caused by increased intracellular levels of proline. Alternative PUT3-dependent and -independent mechanisms might operate in other suppressed strains.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Transportadores de Ácidos Monocarboxílicos/genética , Prolina/metabolismo , Riboflavina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Sitios de Unión , Prueba de Complementación Genética , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Supresión Genética , Transactivadores/metabolismo , Factores de TranscripciónRESUMEN
The riboflavin (vitamin B(2)) biosynthetic genes in Bacillus subtilis are transcribed simultaneously from the riboflavin promoter (P(rib)). The 5'-end of the nascent rib-mRNA carries a flavin mononucleotide (FMN) binding riboswitch, which regulates gene expression. The antibiotic roseoflavin from Streptomyces davawensis is a naturally occurring riboflavin analog, its mechanism of action is largely unknown. A recombinant B. subtilis strain carrying a copy of P(rib)-RFN fused to a promoterless lacZ reporter gene in the chromosomal amyE locus was grown in a minimal medium. Upon addition of roseoflavin to the growth medium the apparent LacZ activity in this strain was not significantly reduced. Similar experiments carried out on recombinant B. subtilis strains oversynthesizing the flavin transporters RibU (B. subtilis) or RibM (S. davawensis) produced still other results. In these strains, roseoflavin (as well as riboflavin) repressed LacZ synthesis indicating that the RFN riboswitch is a target for roseoflavin (or roseoflavin mononucleotide), which may at least in part explain its antibiotic activity.
Asunto(s)
Antibacterianos/biosíntesis , Bacillus subtilis/química , Riboflavina/análogos & derivados , Riboflavina/química , Streptomyces/metabolismo , Antibacterianos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Estructura Molecular , Regiones Promotoras Genéticas , Riboflavina/biosíntesis , Riboflavina/metabolismoRESUMEN
The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Cromatografía de Afinidad/métodos , Clonación Molecular , Modelos Biológicos , Péptidos/química , Proteolípidos/química , Protones , Especificidad por Sustrato , Factores de TiempoAsunto(s)
Membrana Celular/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Análisis de la Célula Individual/métodos , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Transporte Biológico , Ratones , Oocitos/citología , Transportador de Péptidos 1 , Conejos , Simportadores/metabolismo , Xenopus laevisRESUMEN
To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flavoproteínas/metabolismo , Fotorreceptores Microbianos/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Escherichia coli/metabolismo , Flavoproteínas/genética , Mycobacterium tuberculosis/genética , Fotorreceptores Microbianos/genética , Unión Proteica , Riboflavina/análogos & derivados , Riboflavina/genética , Riboflavina/metabolismoRESUMEN
Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.
Asunto(s)
Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Schizosaccharomyces pombe/biosíntesis , Schizosaccharomyces/metabolismo , Simportadores/biosíntesis , Tiamina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antimetabolitos/farmacología , Transporte Biológico/fisiología , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/fisiología , Humanos , Proteínas de Transporte de Membrana/genética , Poliaminas/metabolismo , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Simportadores/genética , Tiamina/genética , Ácido gamma-Aminobutírico/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Riboflavin (vitamin B(2)) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin (K(m) 11 microM). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Corynebacterium glutamicum/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Riboflavina/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Riboflavina/análogos & derivados , Especificidad por Sustrato , Desacopladores/farmacologíaRESUMEN
The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sulfurtransferasas/metabolismo , Factores de Transcripción/metabolismo , Vías Biosintéticas , Biotina/análogos & derivados , Biotina/biosíntesis , Biotina/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/genética , Sulfurtransferasas/biosíntesisRESUMEN
Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.
Asunto(s)
Biotina/química , ADN/genética , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismo , Secuencia de Bases , Biotinilación , Secuencia Conservada , ADN/química , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Mutación , Regiones Promotoras GenéticasRESUMEN
The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlorella/química , Microdominios de Membrana/química , Proteínas de Transporte de Monosacáridos/metabolismo , Simportadores/metabolismo , Chlorella/citología , Detergentes , Ergosterol/metabolismo , Lípidos de la Membrana/química , Proteínas de Transporte de Monosacáridos/análisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Schizosaccharomyces/citología , Esfingolípidos/metabolismo , Simportadores/análisisRESUMEN
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 microM) 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.
Asunto(s)
Proteínas Portadoras/química , Regulación Fúngica de la Expresión Génica , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/fisiología , Riboflavina/farmacocinética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Catálisis , Membrana Celular/metabolismo , Flavina-Adenina Dinucleótido/química , Proteínas Fúngicas , Eliminación de Gen , Genes Fúngicos , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno , Cinética , Modelos Genéticos , Transportadores de Ácidos Monocarboxílicos/química , Mutación , Sistemas de Lectura Abierta , Oxidación-Reducción , Fenotipo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Riboflavina/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Especificidad por Sustrato , Temperatura , Factores de TiempoRESUMEN
Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1+ by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1+ (for vitamin B6 uptake) to replace car1+. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1+ is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride.
Asunto(s)
Amilorida/metabolismo , Diuréticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Piridoxina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Amilorida/química , Amilorida/toxicidad , Diuréticos/química , Prueba de Complementación Genética , Humanos , Proteínas de Transporte de Membrana/genética , Estructura Molecular , Piridoxina/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Especificidad por Sustrato , Tiamina/metabolismoRESUMEN
The fission yeast Schizosaccharomyces pombe is auxotrophic for biotin (vitamin H) and growth depends on biotin uptake over the plasma membrane. Here a biotin transport mutant of Saccharomyces cerevisiae is used to identify the vht1(+) gene encoding the Schizosaccharomyces pombe plasma membrane transport protein for biotin. SpVht1p belongs to the family of allantoate transporters and has only little sequence homology to the S. cerevisiae biotin transporter. Although having dissimilar primary structures, the biotin transporters in Sz. pombe and S. cerevisiae share similar biochemical properties and regulation. Like in S. cerevisiae, biotin uptake in Sz. pombe is a high-affinity process, is optimal at acidic pH values and inhibited by protonophores, indicating that SpVht1p acts as a proton-biotin symporter. Desthiobiotin, the metabolic precursor of biotin, is also imported by SpVht1p. Deletion of vht1(+) abolishes growth on low external concentrations of the vitamin, showing that vht1(+) encodes the only protein that mediates biotin uptake in Sz. pombe. Expression of vht1(+) is maximal at low external biotin concentrations, indicating that Sz. pombe can adjust the rate of biotin uptake to meet the requirement for the vitamin.