FigA,a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae,Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca²⁺ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.     

Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae

Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.     

Kch1 Family Proteins Mediate Essential Responses to Endoplasmic Reticulum Stresses in the Yeasts Saccharomyces cerevisiae and Candida albicans

The activation of a high affinity Ca²⁺ influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K⁺ transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K⁺ and was also dependent on the high affinity K⁺ transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.     

酿酒酵母细胞中钙离子信号传导途径的研究进展

酿酒酵母（SaccharomycescP增v括fdP）细胞可以通过ca2＋／钙调磷酸酶信号途径来应对许多外界环境胁迫。在交配信息素、盐或者其他环境压力存在的条件下,钙离子会通过细胞质膜上的未鉴定的钙转运蛋白x和M或者由Cchl和Midl组成的钙通道进入细胞质。胞质内钙离子浓度的增加会激活细胞质里的钙调磷酸酶（calcineurin）。钙调磷酸酶的一个非常重要的作用是去磷酸化细胞质内的转录因子Crzl,造成它快速地从细胞质转移到细胞核,从而诱导包括液泡膜上钙泵蛋白基因PMCl以及内质网膜和高尔基体膜上钙泵蛋白基因尸脚,在内的目标基因的表达。这两个钙泵蛋白和液泡膜上的Ca2＋／H＋交换蛋白Vcxl一起作用,将细胞质内的钙离子浓度控制在50～200nmol／L的正常生理浓度内．使细胞能够正常生长。该综述主要论述了酿酒酵母细胞内Ca2＋／钙调磷酸酶信号途径的最新研究进展。     

State Transitions in the TORC1 Signaling Pathway and Information Processing in Saccharomyces cerevisiae

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases—Gtr1, Gtr2, and Rho1—bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.     

Uridine Recognition Motifs of Human Equilibrative Nucleoside Transporters 1 and 2 Produced in Saccharomyces cerevisiae

The sugar moiety of nucleosides has been shown to play a major role in permeant‐transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [³H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3′). The C(2′)‐OH at uridine was a structural determinant for uridine‐hENT1, but not for uridine‐hENT2, interactions. Both transporters were sensitive to modifications at C(5′) and hENT2 displayed more tolerance to removal of C(5′)‐OH than hENT1; addition of an O‐methyl group at C(5′) greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3′)‐OH and moderate interactions with C(2′)‐OH and C(5′)‐OH of uridine, whereas hENT2 formed strong interactions with C(3′)‐OH, weak interactions with C(5′)‐OH, and no interaction with C(2′)‐OH.     

Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr¹¹²⁵ and Ser¹¹⁴³. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser¹¹⁴³, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1^T1125 affected the phosphorylation of Pkc1 at Ser¹¹⁴³, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser⁴⁷³. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.     

Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.     

低氧诱导因子-1的转录活性调控及其信号传导

低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)是氧平衡调控相关的转录因子.依赖HIF-1的基因表达调控系统广泛影响葡萄糖代谢、细胞增殖、凋亡和血管发生,与机体低氧适应、胚胎发育、各种缺血性疾病及肿瘤相关.HIF-1自身活性调节是低氧应答基因表达调控的中心环节.调控主要发生在源于Ras的两条信号途径：Ras/Raf/MEK介导的HIF-1反式激活功能调控,PI(3)K/Akt依赖的HIF-1alpha蛋白稳定性调控.这两个信号传导途径分别独立又协调地调控着HIF-1的转录活性.     

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the Yeast Saccharomyces cerevisiae

The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究, 我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因, 并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3为标记基因, 在不含尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株, 该菌株在低K+培养基中导入拟南芥K+转运体基因AtKuP1可恢复正常生长。     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究,我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因,并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3力标记基因,在不舍尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株,该菌株在低K 培养基中导入拟南芥K 转运体基因AtKuP1可恢复正常生长.     

Effect of C-Terminal Protein Tags on Pentitol and l-Arabinose Transport by Ambrosiozyma monospora Lat1 and Lat2 Transporters in Saccharomyces cerevisiae

Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-³H]arabinose, l-[¹⁴C]arabitol, and [¹⁴C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (K_m ≈ 0.03 mM) and l-arabitol and ribitol with very low affinity (K_m ≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (K_ms ≈ 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.     

Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in Saccharomyces cerevisiae Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation

This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae

Metazoan arrestin proteins bind to seven-transmembrane proteins, mediate their internalization and play central roles in the subsequent signal transduction pathway. In Saccharomyces cerevisiae, there are several arrestin-related proteins. One of those proteins, Rod1, has been identified to have the ability to confer resistance to o-dinitrobenzene. We found that Rod1 interacted with Snf4, a subunit of Snf1-kinase complex. Both snf4 and snf1 mutants were also sensitive to the drug and the kinase activity of Snf1 was required for the drug tolerance. In immunoblotting analysis, the Rod1 protein was phosphorylated in an Snf1-dependent manner in vivo, and the phosphorylation of the serine residue 447 of Rod1 was responsible for the band-shift. Furthermore, the Rod1 protein was directly phosphorylated by Snf1-kinase in vitro. The substitution of the serine residue 447 to alanine slightly enhanced the resistance to the drug. We discuss possible functions of Rod1.     

毒蝇碱型乙酰胆碱受体经Ras—ERK—1／2信号通路上调Bcl—2和磷酸化Bad表达

毒蝇碱型乙酰胆碱受体 (muscarinicacetylcholinereceptor,mAChR)和Bcl 2家族蛋白均具有调控神经细胞凋亡和生存的作用 ,然而mAChR和Bcl 2家族蛋白之间的内在联系即信号转导通路仍然不清楚。为此 ,对mAChR调控神经母细胞瘤SH SY5Y细胞生存蛋白Bcl 2和磷酸化Bad的信号转导通路进行了研究。结果显示 :(1)mAChR激动剂卡巴可 (carbachol)不仅活化SH SY5Y细胞的MEK/ERK 1/ 2 ,而且上调Bcl 2和磷酸化Bad的表达 ;(2 )mAChR拮抗剂阿托品、MEK抑制剂PD980 5 9、PKC抑制剂bisindolymaleimide I和Src抑制剂PP1均能完全阻断或显著减弱卡巴可的上述作用 ,但G蛋白脱偶联剂百日咳毒素和PI 3激酶抑制剂wortmannin对卡巴可的上述作用无明显影响 ;(3)显性负突变Ras和Raf均能阻断卡巴可上调转染至SH SY5Y细胞内的Bcl 2启动子的转录调控活性。结果表明 :mAChR通过Gq/ 11、PKC和Src依赖的Ras ERK 1/ 2信号转导通路上调SH SY5Y细胞Bcl 2和磷酸化Bad蛋白表达。这一研究将有助于揭示神经递质、神经营养因子和神经营养药物等抑制神经细胞凋亡、促进神经细胞生存的分子机制。