Assembly and Regulation of the Yeast Vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells. Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V₁) sector, which contains the ATP-binding sites, from the membrane (V₀) sector, which contains the proton pore. A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes. We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure. In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V₁ and V₀, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V₁ sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly.     

光对紫甘蓝花青素合成代谢影响及基因表达模式分析

为了研究光对紫甘蓝花青素合成代谢影响及其调控机制,以“早红”紫甘蓝为试验材料,普通甘蓝“丰园913”（青甘蓝）为对照,对生长1周的幼苗进行遮光处理和光照处理,采用pH差示法测定花青素含量,半定量RT-PCR分析花青素合成途径结构基因表达模式。结果表明：光照处理与遮光处理后,除PAL、UFGT外,紫甘蓝结构基因表达无明显差异,无光条件下,紫甘蓝幼苗仍着色明显,而青甘蓝幼苗完全白化;与青甘蓝相比,紫甘蓝花青素合成途径下游结构基因表达量明显增高,幼苗着色深,揭示紫甘蓝花青素的大量积累与下游结构基因的表达密切相关。     

Subunit C of the Vacuolar-Type ATPase from the Vanadium-Rich Ascidian Ascidia sydneiensis samea Rescued the pH Sensitivity of Yeast vma5 Mutants

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H⁺-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A₁, neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells. Received August 11, 2000; accepted March 5, 2001.     

不同光强光质对艾草生长及挥发油成分积累的影响

为探究光照对艾(Artemisia argyi)的产量及主要挥发油成分积累的影响,以蕲艾组培苗为试材,采用高光强、红光、蓝光等进行不同光强光质处理实验。结果表明,高光强及蓝光处理可促进艾生物量积累,红光和蓝光处理可促进植株伸长。高光强、红光及蓝光均可提高艾叶挥发油中1,8-桉油素、β-萜品醇含量。高光强还能促进龙脑积累,而蓝光处理下可检测到较多在其他光照条件下未检测到的化合物,如萜烯和蓝桉醇等物质。对不同光强光质处理下光受体及光响应转录因子基因表达量进行分析,在高光强处理下,所有光受体的表达量均显著升高;而响应不同光质的对应光受体基因表达量增加。光受体基因表达与艾生物量及主要挥发油的相关性分析表明,光受体基因表达对艾草生物量的影响起重要作用,且不同光处理调控可通过光受体响应并级联调控相关基因表达,调控艾叶生长及挥发油的合成。高光强和蓝光处理下,艾草的生物量及有效挥发油代谢物增加,可在艾草栽培中加以应用。     

Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

种子贮藏蛋白的运输、积累和基因表达调控

种子中贮藏蛋白的运输和积累途径主要有：(1)蛋白质合成后经内膜系统转移到蛋白质贮藏液泡(PSV)中积累;(2)合成的蛋白质直接在粗糙内质网的膜囊中积累形成蛋白质体;(3)贮藏蛋白不经高尔基体的加工由粗糙内质网上合成后直接运输到PSV中积累。贮藏蛋白基因的表达受该基因的顺式作用元件和反式作用因子的共同调控,此外染色体的结构也影响贮藏蛋白基因的表达。     

光、糖与激素影响植物花色素苷合成与积累的研究进展(综述)

次生代谢物质花色素苷存在于植物的叶片、花、果实和种子的表皮细胞的液泡中,是一类使这些器官呈现从红色到黑色等系列颜色的水溶性色素。其合成过程不仅受到基因的调控,还受多种因素影响。首先是光通过信号转导途径直接或间接地调节相关酶基因表达的过程;其次是糖,常与光相互作用协调控制花着色;激素也是影响花色素苷合成的一个重要因素,往往通过影响植物体内的代谢过程和植物基因的表达来影响花色素苷的合成和积累。本文综述近20年来该领域的研究进展。     

葡萄果实成熟期花色苷与白藜芦醇合成相关基因的表达

以酿酒葡萄‘赤霞珠’为试材,采用pH示差法和高效液相色谱(HPLC)法分别测定葡萄成熟期果皮花色苷和白藜芦醇含量,用实时荧光定量PCR检测两者合成途径中相关基因的表达量,分析花色苷含量和白藜芦醇含量与其相关基因表达的关系,以揭示结构基因与调控基因的调控机制,为筛选富含花色苷和白藜芦醇的酿酒葡萄提供理论依据。结果显示:(1)葡萄果皮花色苷含量在花后112d达到最高值(0.77mg/g),反式白藜芦醇含量在花后126d达到最高值(30.87μg/g)。(2)花色苷和白藜芦醇合成途径中,CHSs、CHI、STS、UFGT、MybA1、MybA2基因的表达量除花后98d下调外,其余时间均呈上调表达,而Myb5a则始终呈上调表达。(3)相关分析表明,STS基因表达量与CHS1、CHS2基因表达量呈极显著和显著正相关关系,MybA1、MybA2基因表达量与CHSs、CHI、STS、UFGT基因的表达量呈极显著正相关关系;Myb5a基因表达量与CHS3基因表达量呈极显著正相关关系。研究表明,部分结构基因的表达与花色苷和白藜芦醇的变化不同步,MybA1和MybA2可能调控花色苷合成途径中多个结构基因的表达,花色苷与白藜芦醇的关系并不固定,而是处在动态变化中。     

Concordant Time-Dependent Patterns of Activities and Enzyme Protein Amounts of V-PPase and V-ATPase in induced (Flowering and CAM or Tumour) and Non-Induced Plant Tissues*

There are two H⁺-pumping enzymes at the tonoplast membrane of plant vacuoles, the V-ATPase and the V-PPase. One attempt to explain the enigma of “two H⁺ pumps, one membrane” was the suggestion that the V-PPase has special functions in young developing and growing tissues in utilization of pyrophosphate produced in particularly active metabolism and in pumping of K⁺ for vacuolization. This should lead to reciprocal expression of both enzymes with time during development. Here we used stimulation of Kalanchoë blossfeldiana Poellnitz cv. Tom Thumb plants by short-day treatments to induce crassulacean acid metabolism and flowering and of Ricinus communis L. stem tissue by infection with Agrobacterium tumefaciens strain C58 to induce vigorous growth of tumours, and we compared these stimulated tissues with leaves of non-stimulated long-day controls and non-infected stem tissue, respectively. Activities and protein levels of both enzymes increased (K. blossfeldiana) or remained high (R. communis) in the stimulated tissues and decreased in the non-stimulated tissues with time. Time-dependent patterns of the two enzymes were concordant in all of the four cases and not inverse, i.e. two plants with two different conditions each, leading to very different developmental situations.     

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.     

光照和温度对豌豆Lhcb2基因表达的影响

采用ＲＴ－ＰＣＲ技术,从豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）幼叶中克隆了１个约８００ｂｐ的Ｌｈｃｂ２ ｃＤＮＡ。以特异探针进行的Ｓｏｕｔｈｅｒｎ杂交结果表明,Ｌｈｃｂ２基因以单拷贝形成存在于豌豆基因组中。不同光照时间和温度对豌豆幼苗进行处理的ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ ｂｌｏｔｔｉｎｇ分析表明,Ｌｈｃｂ２基因转录水平上的表达受光照的控制,且明显表现出对光照时间的依赖性。光照０￣１．５ｈＬｈ     

叶片发育影响紫罗勒花青素的强光诱导和激发能分配

通过气体交换、叶绿素荧光、反射光谱和显微技术等研究了叶片发育与花青素强光诱导的关系及其对激发能分配的影响。结果表明,遮荫导致紫罗勒叶片变薄,花青素含量显著降低。当弱光下生长的植株转入强光后,转光前发育成熟的叶片花青素含量很低,而此后强光下发育成熟的叶片花青素含量高。转强光后,弱光下发育成熟的叶片光合速率低、光抑制严重,且天线耗散增强;强光下发育成熟的叶片净光合速率高,光抑制程度轻,天线耗散低。因此,我们认为叶片发育影响紫罗勒花青素合成的强光诱导,而转强光后花青素的诱导差异进一步改变了光合作用过程中的激发能分配。     

Enzymatic Improvement of Food Flavor I. Purification and Characterization of Bovine Liver Mitochondrial Aldehyde Dehydrogenase

Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215,000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54,000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1 µm, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.     

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V₁) responsible for hydrolysis of ATP and an integral domain (V₀) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V₁ and V₀ domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.     

心里美萝卜花青素合成酶基因RsANS克隆及花青素生物合成相关基因表达分析

花青素合成酶(ANS,anthocyanidin synthase)是植物花青素合成的关键酶,催化无色花色素转变成有色花色素。本研究从心里美萝卜HX12Q-49中克隆获得了花青素合成酶基因Rs ANS(Gen Bank登录号:KR262954)。该基因全长1137 bp,包含2个外显子和1个内含子;开放阅读框1071 bp,编码356个氨基酸。同源性分析显示Rs ANS蛋白与大白菜、甘蓝和芥菜ANS蛋白同源性较高。qRT-PCR表明Rs ANS在心里美萝卜5个不同发育时期均有表达,且在破肚期表达量最高。通过对花青素合成途径其他8个结构基因和3个调控基因表达进行分析,结果显示心里美萝卜中b HLH转录因子在不同发育时期的表达模式与Rs ANS、CHI、DFR和UFGT基本一致,即在破肚期的表达量最高;WD40转录因子的表达与CHS类似,其表达量在芽期、破肚期、膨大前期和膨大盛期逐渐上升,随后在成熟期下降。     

观赏羽衣甘蓝BoDFR基因的克隆及表达分析

为揭示羽衣甘蓝二氢黄酮醇4 还原酶 (DFR)基因调控花青素合成的功能,该研究对不同叶色羽衣甘蓝的叶片花青素含量进行测定,根据结球甘蓝DFR序列信息,利用 RT PCR 技术克隆羽衣甘蓝BoDFR基因并进行实时荧光定量表达分析。结果表明: BoDFR的cDNA全长为1 158 bp,编码385个氨基酸,其蛋白相对分子质量为42 925.06 Da,预测亚细胞定位为细胞质;蛋白质二级结构分析表明α 螺旋和无规则卷曲为DFR 蛋白的主要二级结构元件。序列比对显示 DFR 蛋白具有 NADPH 结合位点和底物结合位点,属于NADB Rossmann 超基因家族。系统进化分析表明,BoDFR与结球甘蓝(Brassica oleracea var. capitata)DFR亲缘关系最近。花青素含量测定显示,紫叶羽衣甘蓝叶片中花青素含量最高,粉叶羽衣甘蓝含量较高,而白叶羽衣甘蓝叶片中检测不到花青素。实时荧光定量 PCR 分析表明,BoDFR基因表达量与花青素含量高低一致,其中紫叶羽衣甘蓝叶片中BoDFR基因的表达量最高,而白叶羽衣甘蓝心叶中仅微量表达。     

红肉葡萄果实发育过程中花青苷组分变化与基因表达规律分析

以红肉葡萄新种质‘钟山红玉’为实验材料,通过高效液相色谱-质谱联用(HPLC-MS)检测‘钟山红玉’从幼果到成熟期果皮、果肉中的花青苷组分及含量,并利用实时荧光定量PCR检测不同发育时期花青苷合成相关基因的表达水平,研究其不同果实发育时期果皮、果肉中的花青苷组分变化,以及相关基因表达规律,探索红肉葡萄果实呈色机制,为葡萄果实品质改良育种提供理论依据。结果表明:(1)‘钟山红玉’果皮和果肉中共检测出13种花青苷且都为单糖苷花青苷,与欧亚种葡萄亲缘关系较近。(2)‘钟山红玉’果皮、果肉中均检测出了欧亚种葡萄中很少有的天竺葵素3-O-葡萄糖苷;果皮中飞燕草素类(飞燕草素-、锦葵色素-、矮牵牛素-)花青苷衍生物含量显著高于矢车菊素类(矢车菊素-、芍药素-)花青苷衍生物,而在果肉中则两大类花青苷含量相近,这与果皮中较高的F3′5′H基因表达量有关,且在果皮中酰基化花青苷比例显著高于果肉。(3)花青苷合成相关基因MYBA2和UFGT在‘钟山红玉’不同发育时期的表达变化规律一致,UFGT基因在果肉中基本不表达,而MYBA2可能是决定‘钟山红玉’果肉转色的关键因子;并且ABA响应相关基因PYL1在‘钟山红玉’发育时期的表达规律与OMT和LDOX基因一致。     

Localization of Pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V-ATPase activities. By immunogold labelling we have shown that H⁺-pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V-ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V-ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 ? 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,-head of V-ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate-sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely-packed 6 ? 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5- and 6-fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V₀ body of the V-ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.