Glucose controls phosphoregulation of hydroxymethylglutaryl coenzyme A reductase through the protein phosphatase 2A-related phosphatase protein, Ppe1, and Insig in fission yeast

HMG-CoA reductase (HMGR) catalyzes a rate-limiting step in sterol biosynthesis and is a key control point in the feedback inhibition that regulates this pathway. Through the action of the membrane protein Insig, HMGR synthesis and degradation are regulated to maintain sterol homeostasis. The fission yeast Schizosaccharomyces pombe encodes homologs of HMGR and Insig called hmg1(+) and ins1(+), respectively. In contrast to the mammalian system, Ins1 regulates Hmg1 by a nondegradative mechanism involving phosphorylation of the Hmg1 active site. Here, we investigate the role of the Ins1-Hmg1 system in coupling glucose sensing to regulation of sterol biosynthesis. We show that Ins1-dependent Hmg1 phosphorylation is strongly induced in response to glucose withdrawal and that HMGR activity is correspondingly reduced. We also find that inability to activate Hmg1 phosphorylation under nutrient limiting conditions results in overaccumulation of sterol pathway intermediates. Furthermore, we show that regulation of Hmg1 phosphorylation requires the protein phosphatase 2A-related phosphatase Ppe1 and its regulator Sds23. These results describe a mechanism by which cells tune the rate of sterol synthesis to match nutrient availability.     

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.     

Isolation of the ribosomal protein gene S6 from Schizosaccharomyces pombe

Summary We screened a Schizosaccharomyces pombe genomic library using the ribosomal protein gene SI0 from Saccharomyces cerevisiae as a probe. Hybrid-selected translation of the positive clones revealed a ribosomal protein of S. pombe which is probably equivalent to the ribosomal protein SI0 from S. cerevisiae.     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

Conserved and unique features of the fission yeast core Atg1 complex

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.     

茶树HMG-CoA还原酶基因全长cDNA克隆及序列分析

香气是茶叶的重要品质之一,萜类物质不仅香气好,而且沸点普遍较高,是构成茶叶香气的重要物质基础,决定着茶叶的香气品质,也可作为茶叶香型划分的依据。在植物中,倍半萜、多萜醇等通过胞质中的甲瓦龙酸(MVA)途径合成。HMG-Co A还原酶(HMGR)催化HMG-Co A(3-羟基-3-甲基戊二酸单酰辅酶A)生成甲瓦龙酸,是依赖MVA萜类合成途径的关键限速反应。为了有助于理解茶树萜类合成的分子遗传机制,通过RACE-PCR方法从茶树中克隆了一个编码HMG-Co A还原酶的c DNA全长序列(命名为Cs HMGR1),该序列由1 979 bp组成,包含一个1 722 bp的完整开放阅读框,编码573个氨基酸。其推定的编码蛋白与橡胶树、旱莲木、人参、荔枝、西洋参、丹参、罗汉果及龙眼的同源蛋白具有80%~82%的序列一致性。利用Cs HMGR1和其它物种HMGR同源蛋白的催化区域构建系统发育树,表明其属于真核生物I类HMGR家族。结构分析表明,Cs HMGR1含有两个跨膜区,推测其与其它真核生物同源蛋白类似地定位于内质网上;含有两个HMG-Co A结合位点、两个NADPH结合位点、四个保守的催化活性残基及一个磷酸化位点,说明磷酸化/去磷酸化很可能也是其活性调节的重要方式。表达分析表明,Cs HMGR1在"大叶龙"叶芽、母株叶芽及花芽都有较强的表达。其表达调控及生理活性对茶叶品质可能有重要影响,并在其功能解析的基础上,有可能作为茶叶品质鉴定及育种的一个依据。     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

S-phase and DNA-damage checkpoints: a tale of two yeasts

Many genes required for the S-phase and DNA-damage checkpoints have been identified in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This year many checkpoint genes have been sequenced, providing new information about the mechanism of checkpoint control. Several of these genes are conserved between the two yeasts but others are species-specific.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.     

Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase

Summary The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34^cdc2 contains no phosphoserine.     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha

Summary The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 by intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.     

Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Centromere localization and function of Mis18 requires Yippee‐like domain‐mediated oligomerization

Mis18 is a key regulator responsible for the centromere localization of the CENP‐A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP‐A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N‐terminal Yippee‐like domain, is crucial for its centromere localization and function. The crystal structure of the N‐terminal Yippee‐like domain reveals a fold containing a cradle‐shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N‐terminal Yippee‐like domain forms a homodimer in vitro and in vivo, full‐length Mis18, including the C‐terminal α‐helical domain, forms a homotetramer in vitro. We also show that the Yippee‐like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.