Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae

Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.     

Differential regulation and substrate preferences in two peptide transporters of Saccharomyces cerevisiae

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [(14)C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [(14)C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.     

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.     

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.     

Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus.

The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpTLH) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. Functional expression of the peptide transporter was shown by the uptake of prolyl-[14C] alanine in whole cells and membrane vesicles. Peptide transport via DtpT in membrane vesicles is driven by the proton motive force. The system has specificity for di- and tripeptides but not for amino acids or tetrapeptides. The dtpTLH gene consists of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpTLH shows 34% identity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin, but the similarity between these proteins is confined mainly to the N-terminal halves.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12 - 14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.     

Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.     

Ssy1p and Ptr3p Are Plasma Membrane Components of a Yeast System That Senses Extracellular Amino Acids

Mutations in SSY1 and PTR3 were identified in a genetic selection for components required for the proper uptake and compartmentalization of histidine in Saccharomyces cerevisiae. Ssy1p is a unique member of the amino acid permease gene family, and Ptr3p is predicted to be a hydrophilic protein that lacks known functional homologs. Both Ssy1p and Ptr3p have previously been implicated in relaying signals regarding the presence of extracellular amino acids. We have found that ssy1 and ptr3 mutants belong to the same epistasis group; single and ssy1 ptr3 double-mutant strains exhibit indistinguishable phenotypes. Mutations in these genes cause the nitrogen-regulated general amino acid permease gene (GAP1) to be abnormally expressed and block the nonspecific induction of arginase (CAR1) and the peptide transporter (PTR2). ssy1 and ptr3 mutations manifest identical differential effects on the functional expression of multiple specific amino acid transporters. ssy1 and ptr3 mutants have increased vacuolar pools of histidine and arginine and exhibit altered cell growth morphologies accompanied by exaggerated invasive growth. Subcellular fractionation experiments reveal that both Ssy1p and Ptr3p are localized to the plasma membrane (PM). Ssy1p requires the endoplasmic reticulum protein Shr3p, the amino acid permease-specific packaging chaperonin, to reach the PM, whereas Ptr3p does not. These findings suggest that Ssy1p and Ptr3p function in the PM as components of a sensor of extracellular amino acids.     

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

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.     

高山离子芥铁转运蛋白基因编码序列的克隆及其抗逆性表达的实时定量分析

铁转运蛋白 (iron transport protein,IRT)是具有跨膜运输离子功能的特殊蛋白质,属于金属离子转运家族的成员．本研究运用RACE方法从高山离子芥（Chorispora bungeana）中克隆得到完整的铁转运蛋白cDNA,命名为CbIRT（基因登录号为EU330924）．该基因全长1 290 bp,包含1个1 035 bp的开放阅读框（ORF）,编码344个氨基酸的蛋白．系统进化树分析显示,该基因与拟南芥AtIRT1和遏蓝菜TcIRT-G的亲缘关系最近,同源性分别达到了87.4%和86.5%,而在氨基酸序列水平与拟南芥AtIRT1的同源性达到了89%,表明克隆得到的CbIRT属于金属离子转运体家族成员．实时荧光定量方法对高山离子芥CbIRT基因在不同温度和铁营养水平条件下的表达情况进行分析表明,CbIRT对零下低温和零上低温的表达水平呈截然不同的反应;正常铁营养状态下,CbIRT是微量表达的,而缺铁及低温处理都可以大幅度地促进该基因的表达,富铁可以抑制该基因的表达．显示了该蛋白在转运Fe离子方面的重要作用．     

Use of the signal peptide of Pisum vicilin to translocate beta-glucuronidase in Nicotiana tabacum.

A hybrid protein system was used for the study of protein transport in plant cells. A nucleotide sequence (vic) encoding a putative signal peptide of 15 amino acid residues, derived from the published aa sequence of one Pisum vicilin, was synthesized and fused in frame to the gus gene encoding a bacterial cytosolic beta-glucuronidase (GUS). When the hybrid vic::gus gene was expressed in tobacco cells using the cauliflower mosaic virus 35S promoter, the hybrid GUS protein was targeted to, and glycosylated inside the rough endoplasmic reticulum. Glycosylation could be blocked with the antibiotic tunicamycin. The study of transient expression in protoplasts showed that extracellular secretion efficiency was low, which may be due to the nature of the GUS protein.     

人胰岛素原类似物(BKRA)基因的合成与表达

为了利用基因工程生产胰岛素,按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因,其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解,通过人工接头将２个ＢＫＲＡ基因串联起来,接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋）,实现了在大肠杆菌中的融合表达,表达产物以包含体形式存在,约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段,以ＨｉＴｒａｐ凝胶进行亲和层析,一步纯化可达纯度９５％以上．放射免疫测定表明,纯化的融合蛋白具有胰岛素抗原活性．表明已构建成人胰岛素原类似物的高效表达菌株