Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae

BACKGROUND INFORMATION: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail. RESULTS AND CONCLUSIONS: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.     

Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

酿酒酵母Afr1p调控septin蛋白Cdc12p的定位

AFR1最初被鉴定为在过量表达的情况下,可以使细胞产生α因子抗性,同时对融合过程中融合突起的形成起重要作用。Afr1p还具有调节细胞壁完整性途径中的MAPK Mpk1p的定位及活性的功能。该文通过对蛋白定位的观察发现,半乳糖诱导GAL-AFR1过量表达破坏了在出芽过程中Cdc12p的定位;缺失AFR1也会导致Cdc12p定位异常。Western blot结果显示,在营养生长过程中Afr1p稳定表达。这说明,稳定表达的AFR1有调节septin Cdc12p定位的功能,从而对维持septin的结构起到一定的作用。     

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.     

人工酵母后鲨烯路径基因对7-脱氢胆固醇合成的影响

酵母内源后鲨烯路径中的固醇类物质,是异源甾体类药物合成的重要前体。为了通过微调后鲨烯路径,与异源模块进行适配,以期达到提高异源甾体类化合物表达的目的,以维生素D₃的直接前体-7-脱氢胆固醇(7-DHC)的合成为例,首先在固醇C-24甲基转移酶(ERG6)缺失的酿酒酵母BY4742中,通过导入人源固醇C-24 还原酶DHCR24,并过表达截短的羟甲基戊二酰辅酶A还原酶tHMGR,获得可以合成7-DHC的人工酵母。在此基础上,将后鲨烯路径分割并构建成ERG1、ERG7、ERG11、ERG24-25-26-27和ERG2-3这5个模块,分别在所构建的7-DHC合成菌株中过表达。通过GC-TOF/MS分析7-DHC以及后鲨烯路径中相关代谢中间体的含量,并结合主成分分析发现,过表达不同后鲨烯模块会引起后鲨烯路径上固醇组分的变化而最终影响7-DHC的产量:与出发菌株相比,过表达ERG11模块会显著强化其他固醇物质到酵母固醇的转化;而过表达ERG2-3模块则会减少鲨烯的积累,同时显著增加羊毛固醇及其之后的固醇组分的含量,并获得迄今为止7-DHC在微生物中摇瓶水平的最高产量。因此,对ERG11和ERG2-3的表达优化对7-DHC的合成以及后鲨烯路径代谢流的强化起到了显著的作用,是后续优化人工7-DHC合成酵母的潜在靶点。为研究后鲨烯路径与其他异源甾体合成模块间的适配,提供了可供参考的案例。     

酿酒酵母促分裂原蛋白激酶Hog1p 介导的渗透胁迫反应调控机制

 高渗透性甘油促分裂原激酶信号转导途径（high osmolarity glycerol mitogen activated protein kinase signaling transduction pathway,HOG-MAPK）是调控酿酒酵母对外界高渗透压胁迫环境应答的主要途径,促分裂原蛋白激酶Hog1p（MAPK Hog1p）是其中的关键性作用因子.在高渗透压刺激时,MAPK Hog1p接受信号被特异性激活并进入核内,调控相关胁迫应答基因的表达,并介导该时期细胞周期的阻滞,从而增强细胞对外界不利环境的适应能力.对胁迫条件下酿酒酵母中MAPK Hog1p作用机制的进一步研究,有利于更深入地了解哺乳动物体内逆境激发促分裂原蛋白激酶途径的功能和调控机制.     

Catabolite inactivation of the sugar transporters in Saccharomyces cerevisiae is inhibited by the presence of a nitrogen source

Saccharomyces cerevisiae uses glucose preferentially to any other carbon source and this preferential use is ensured by control mechanisms triggered by glucose. The consensus is that inactivation of sugar transporters other than glucose transporters is one of these mechanisms. This inactivation is called catabolite inactivation because of its apparent analogy with the catabolite inactivation of gluconeogenic enzymes. Recently, doubt has been cast on the role of the inactivation of the sugar transporters in controlling the use of glucose because this inactivation neither is specifically triggered by glucose nor specifically affects non-glucose sugar transporters. Based on the fact that this inactivation has been almost exclusively investigated using nitrogen-starved cells, it has been proposed that it might be due to the stimulation of the protein turnover that follows nitrogen starvation. The results obtained in this work support this possibility since they show that the presence of a nitrogen source in the medium strongly inhibited the inactivation. It is concluded that, in growing yeast cells, the contribution of the inactivation by glucose of the non-glucose sugar transporters to the preferential use of glucose is much lower than generally believed.     

The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae

Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.     

Substrate preference is altered by mutations in the fifth transmembrane domain of Ptr2p,the di/tri-peptide transporter of Saccharomyces cerevisiae

The integral membrane protein Ptr2p transports di/tri-peptides into the yeast Saccharomyces cerevisiae. The sequence FYXXINXG (FYING motif) in the 5th transmembrane domain (TM5) is invariably conserved among the members of the PTR (Peptide TRansport) family ranging from yeast to human. To test the role of TM5 in Ptr2p function, Ala-scanning mutagenesis of the 22 residues comprising TM5 was completed. All mutated transporters, with the exception of the Y248A mutant, were expressed as determined by immunoblots. In peptide-dependent growth assays, ten mutants of the non-FYING residues grew as well as wild-type Ptr2p on all twelve different peptides tested. All of the FYING motif mutants, except the non-expressed Y248A, plus seven other mutants in TM5 exhibited differential growth on peptides including Leu-Leu and Met-Met-Met indicating that these mutations conferred substrate preference. In assays measuring direct uptake of the radioactive peptides ³H-Leu-Leu or ¹⁴C-Met-Met-Met, the F, I and G mutants of the FYING motif did not demonstrate accumulation of these peptides over a ten minute interval. The mutation N252A of the FYING motif, along with L240A, M250A, and L258A, exhibited differential substrate preference for Met-Met-Met over Leu-Leu. Other mutations (T239A, Q241A, N242A, M245A, and A260) resulted in preference for Leu-Leu over Met-Met-Met. These data demonstrate that TM5, in particular its conserved FYING motif, is involved in substrate preference of Ptr2p.     

铅和硒对端粒长度、端粒酶及端粒结合蛋白的影响

以酿酒酵母细胞为实验材料 ,在分子水平上研究铅 (Pb)和硒 (Se)对端粒长度、端粒酶及端粒结合蛋白的影响。结果发现 :与对照组相比 ,添加 1mg/LPb的培养基中培养 10 0代后的酿酒酵母细胞中端粒DNA的平均长度缩短 ,端粒结合蛋白Rap1p含量减少 ,而且Rap1p蛋白的二级结构受到扰动、端粒酶活性降低、43%的细胞死亡。加 1mg/LSe培养 10 0代后的酿酒酵母细胞与对照组相比 ,细胞中端粒平均长度增加 ,Rap1p蛋白浓度和二级结构保持稳定 ,端粒酶活性增加 ,细胞正常存活。以上结果表明 ,Pb对酿酒酵母细胞端粒有损伤 ,而且其损伤在子代细胞中有累积效应 ;而Se对Pb损伤具有一定程度的修复保护作用 ,适量给机体补Se对抑制细胞损伤和衰老有一定作用。由于端粒的特殊结构特征 ,推断Pb和Se是通过作用于端粒酶及端粒结合蛋白而间接影响端粒长度的     

代谢工程改造酿酒酵母生产L-乳酸的研究进展

L-乳酸是一种重要的有机化合物,具有广泛的应用价值。微生物发酵法生产是当前L-乳酸的主要来源,但受限于精确的发酵条件、菌体产物耐受能力低及底物要求高等因素,导致L-乳酸供给不足且价格偏高。鉴于酿酒酵母利用廉价底物生产有价值物质方面的诸多优势,并随着分子生物学技术的发展,利用代谢工程改造酿酒酵母本身固有的代谢网络,使其高产L-乳酸已成为当前研究的热点。从L-乳酸的异源生产、关键途径改造及菌体生长能力恢复三个方面归纳了关于代谢工程改造酿酒酵母生产L-乳酸的研究进展。最后,指出了酿酒酵母异源生产L-乳酸存在的不足和今后研究的方向。     

A new function of isonitrile as an inhibitor of the Pdr5p multidrug ABC transporter in Saccharomyces cerevisiae

Pdr5p in Saccharomyces cerevisiae is a functional homologue of mammalian P-glycoprotein implicated in multidrug resistance (MDR). In order to obtain useful inhibitors to overcome MDR in clinical tumors, screening of Pdr5p inhibitors has been carried out. We isolated a fungal strain producing Pdr5p inhibitors using our original assay system, and it was classified as Trichoderma sp. P24-3. The purified inhibitor was identified as isonitrile, 3-(3'-isocyano-cyclopent-2'-enylidene)-propionic acid, a compound whose carboxyl residue is essential for the inhibitory activity. A non-toxic concentration of the isonitrile (41.5 microg/ml, 255 microM) inhibited Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. In addition, addition of the isonitrile led to accumulation of rhodamine 6G, a substrate of Pdr5p, in the Pdr5p-overexpressing cells. The inhibitory profiles of the isonitrile against S1360 mutants (S1360A and S1360F) of Pdr5p were different from those of FK506 and enniatin. The isonitrile did not influence PDR5 gene expression and the amount of Pdr5 protein, nor did it inhibit the function of Snq2p, a homologue of Pdr5p. Interestingly, the isonitrile inhibited the function of Cdr1p and Cdr2p, Pdr5p homologues in pathogenic yeast Candida albicans. Thus, it was found that the isonitrile shows a different inhibitory spectrum from that of FK506 and enniatin as a potent inhibitor for Pdr5p, Cdr1p, and Cdr2p.     

A design for the control of apoptosis in genetically modified Saccharomyces cerevisiae

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.     

Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae

We have developed a new heterologous expression system for monocarboxylate transporters. The system is based on a Saccharomyces cerevisiae pyk1 mae1 jen1 triple-deletion strain that is auxotrophic for pyruvate and deficient in monocarboxylate uptake. Growth of the yeast cells on ethanol medium supplemented with pyruvate or lactate was dependent on the expression of a suitable monocarboxylate transporter. We have used the system to characterize the functional significance of interactions between the rat MCT1 transporter and its ancillary protein CD147. CD147 was shown to improve trafficking of MCT1 to the plasma membrane and its uptake activity. Our results demonstrate a new strategy for the production of properly folded and correctly targeted membrane proteins in a microbial expression system by co-expression of appropriate accessory proteins.     

Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae

Monoterpenes are a diverse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β‐pinene, limonene, myrcene, γ‐terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two‐phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100‐fold increase in the MIC compared to the monoterpenes in a solvent‐free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702‐fold (308 mM, 42.1 g L^?1 of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet‐A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co‐product and extractant for microbial terpene‐based jet fuel production in a two‐phase system offers an attractive bioprocessing option. Biotechnol. Bioeng. 2012; 109: 2513–2522. © 2012 Wiley Periodicals, Inc.