截形苜蓿液泡膜H^＋ -PPase基因克隆与序列分析

采用EST电子克隆技术,以拟南芥AVP1基因cDNA序列为信息探针,在GenBank中对豆科模式植物截形苜蓿(Medicago truncatula)的同源EST序列进行查询比较和拼接,获得了1个完整的cDNA序列跨叠群(contig),并通过RT-PCR成功获得了该cDNA序列,将其命名为MtVP1.该序列包含1个2 298 bp的最大读码框,编码765个氨基酸.MtVP1编码的氨基酸序列与来自绿豆、拟南芥等高等植物的Ⅰ类液泡膜H+-PPase的氨基酸序列的一致性高达84%～93%,疏水性分析和结构预测表明MtVP1编码蛋白是一个典型的膜蛋白,含有13个跨膜区,含有3个在液泡膜H+-PPase中高度保守的区域(CS1、CS2和CS3).从结构分析结果推测MtVP1与AVP1在功能上具有相似性.     

盐爪爪Na^＋/H^＋逆向转运蛋白和焦磷酸酶的亚细胞定位

将盐爪爪Na+/H+逆向转运蛋白基因(KfNHX1)和焦磷酸酶基因(KfVP1)分别构建至植物表达载体,利用基因枪介导的方法转化洋葱表皮细胞,通过荧光显微镜观察研究其亚细胞定位.结果表明,转化了KfNHX1(或KfVP1)-GFP融合蛋白的洋葱表皮细胞仅膜系统散发荧光,而对照组即未转入KfNHX1(或KfVP1)基因的细胞则整体均匀发出荧光.说明KfNHX1和KfVP1可能定位于细胞的膜系统,作为跨膜转运蛋白在离子的调控运输中发挥重要作用.     

A Conserved Domain Is Crucial for Acceptor Substrate Binding in a Family of Glucosyltransferases

Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis. Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP from S. agalactiae strain COH1, has been implicated in bacterial virulence. Four genes (gtfA, gtfB, gtfC, and gtfD) located downstream of srr2 share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously that gtfA and gtfB encode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.     

百脉根液泡膜H+-PPase基因的cDNA克隆与分析

采用EST电子克隆和RACE技术从豆科模式植物百脉根中克隆到一个液泡膜H -PPase基因的cDNA,命名为LcVP1。该cDNA长为2962bp,含2304bp的完整开放阅读框,编码767个氨基酸,其推测的氨基酸序列与绿豆、拟南芥等I类液泡膜H -PPase的氨基酸序列同源性在80%以上,且有很高的功能区段保守性。该cDNA序列已提交GenBank,登录号为EF440187。半定量RT-PCR表明,LcVP1在根、茎、叶中的表达不同,叶中表达最多,茎中最少。     

等渗盐胁迫对番茄抗氧化酶和ATP酶及焦磷酸酶活性的影响

用Ca(NO3)2 80 mmol/L和NaCl 120 mmol/L等渗溶液处理番茄幼苗后,细胞质和叶绿体中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)的活性升高,并且NaCl胁迫的作用明显高于Ca(NO3)2胁迫.Ca(NO3)2处理提高了线粒体中SOD、CAT、APX的活性,而NaCl处理降低了它们的活性.根系质膜H -ATPase、液泡膜H -ATPase、焦磷酸酶(H -PPase)的活性和叶片丙二醛(MDA)及脯氨酸含量在两种盐胁迫后明显增加.NaCl处理对植株生长的抑制程度明显高于Ca(NO3)2处理.     

Mutational Analysis of CvaA in the Highly Conserved Domain of the Membrane Fusion Protein Family

The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue–hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV. Received: 6 February 1999 / Accepted: 26 June 1999     

Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H⁺-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen, and imply the biological significance of the V-PPase in pollen maturation.     

Role of Conserved Residues of the WRKY Domain in the DNA-binding of Tobacco WRKY Family Proteins

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn²⁺. Substitution of the conserved cysteine and histidine residues of the plant-specific C₂H₂-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.     

外源多胺对低氧胁迫下黄瓜幼苗根系生长及H+-ATP酶和H+-焦磷酸酶活性的影响

采用营养液水培方式,研究了根际低氧胁迫下外源多胺对黄瓜幼苗植株根系生长,内源多胺含量与质膜H -ATP酶、液泡膜H -ATP酶和焦磷酸酶活性的影响.结果表明,根际低氧胁迫显著抑制黄瓜幼苗根系的生长,外源Put(腐胺)和Spd(亚精胺)可缓解低氧胁迫对根系的生长抑制,多胺主要以Spd的形式发挥促进性的生理作用,Put通过转化为Spd发挥作用;低氧胁迫下黄瓜根系内源多胺含量略有提高,外源多胺处理可增加内源多胺的含量;低氧胁迫下外源Put和Spd处理后质膜H -ATP酶活性显著提高,外源多胺对黄瓜根系液胞膜H -ATP酶和H -焦磷酸酶活性没有明显影响,说明低氧胁迫下外源多胺主要通过提高质膜H -ATP酶活性而发挥生理作用.     

Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na⁺/H⁺exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na⁺/H⁺ exchanger.     

Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots

The effects of polyamines (Putrescine— Put; Spermidine—Spd; and Spermine—Spm) on␣salt tolerance of seedlings of two barley (Hordeum vulgare L.) cultivars (J4, salt-tolerant; KP7, salt-sensitive) were investigated. The results showed that, the salt-tolerant cultivar J4 seedlings accumulated much higher levels of Spd and Spm and lower Put than the salt-sensitive cultivar KP7␣under salt stress. At the same time, the dry weight of KP7 decreased significantly than that of␣J4. After methylglyoxal bis(guanylhydrazone) [MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC)] treatment, Spd and Spm levels together with the dry weight of both cultivars were reduced, but the salt-caused dry weight reduction in two cultivars could be reversed by the concomitant treatment with Spd. MGBG decreased the activities of tonoplast H⁺-ATPase and H⁺-PPase too, but the experiments in vitro indicated that MGBG was not able to affect the above two enzyme activities. However, the polyamines, especially Spd, promoted their activities obviously. These results suggested that the conversion of Put to Spd and Spm and maintenance of higher levels of Spd and Spm were necessary for plant salt tolerance.     

Palmitoylation of Neurofascin at a Site in the Membrane-Spanning Domain Highly Conserved Among the L1 Family of Cell Adhesion Molecules

Abstract: This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.     

Isolation and Characterization of a Mo6+ -Reducing Bacterium

A Mo⁶⁺ -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo⁶⁺, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo⁶⁺ (10 mM), the bacterium reduced Mo⁶⁺ to form molybdenum blue. Approximately 27% of Mo⁶⁺ added to the medium was reduced after 28 h of cultivation. The reduction of Mo⁶⁺ with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo⁶⁺ reduction. NADH and N,N,N′,N′ -tetramethyl-p-phenylenediamine served as electron donors for Mo⁶⁺ reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo⁶⁺ reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo⁶⁺ reduction. Both ferric and stannous ions strongly enhanced the activity of Mo⁶⁺ reduction by NADH.     

Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide

Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2003 as a significant threat to human health, and CoVs still represent a leading source of novel viruses for emergence into the human population. The CoV spike (S) protein mediates both receptor binding (via the S1 domain) and membrane fusion (via the S2 domain) and shows many features of a class I fusion protein, including the presence of distinct heptad repeats within the fusion domain (37). A critical feature of any viral fusion protein is the so-called “fusion peptide,” which is a relatively apolar region of 15 to 25 amino acids that interacts with membranes and drives the fusion reaction (9, 34, 38). Fusion peptides can be classified as N-terminal or internal, depending on their location relative to the cleavage site of the virus fusion protein (23). One key feature of viral fusion peptides is that within a particular virus family, there is high conservation of amino acid residues; however, there is little similarity between fusion peptides of different virus families (26). Despite these differences, some common themes do emerge, including a high level of glycine and/or alanine residues, as well as critical bulky hydrophobic amino acids. In several cases, the fusion peptide is known to contain a central “kink.” In the case of influenza virus hemagglutinin (HA), which is a classic example of an N-terminal fusion peptide, the N- and C-terminal parts of the fusion peptide (which are α-helical) penetrate the outer leaflet of the target membrane, with the kink at the phospholipid surface. The inside of the kink contains hydrophobic amino acids, with charged residues on the outer face (18). Internal fusion peptides (such as Ebola virus [EBOV] GP) often contain a conserved proline near their centers but also require a mixture of hydrophobic and flexible residues similar to N-terminal fusion peptides (9, 11). It is believed that the kinked fusion peptide sits in the outer leaflet of the target membrane and possibly induces positive curvature to drive the fusion reaction (22). It is important to note that, despite the presence of key hydrophobic residues, viral fusion peptides often do not display extensive stretches of hydrophobicity and can contain one or more charged residues (8). Ultimately, fusion peptide identification must rely on an often complex set of criteria, including structures of the fusion protein in different conformations, biophysical measurements of peptide function in model membranes, and biological activity in the context of virus particles.To date, the exact location and sequence of the CoV fusion peptide are not known (4); however, by analogy with other class I viral fusion proteins, it is predicted to be in the S2 domain. Overall, three membranotropic regions in SARS-CoV S2 have been suggested as potential fusion peptides (14, 17). Based on sequence analysis and a hydrophobicity analysis of the S protein using the Wimley-White (WW) interfacial hydrophobic interface scale, initial indications were that the SARS-CoV fusion peptide resided in the N-terminal part of HR1 (heptad repeat 1) (5, 6), which is conserved across the Coronaviridae. Mutagenesis of this predicted fusion peptide inhibited fusion in syncytia assays of S-expressing cells (28). This region of SARS-CoV has also been analyzed by other groups in biochemical assays (16, 17, 29) and defined as the WW II region although Sainz et al. (29) actually identified another, less conserved and less hydrophobic, region (WW I) as being more important for fusion. Peptides corresponding to this region have also been studied in biochemical assays by other groups (13). In addition, a third, aromatic region adjacent to the transmembrane domain (the membrane-proximal domain) has been shown to be important in SARS-CoV fusion (15, 20, 25, 30). This membrane-proximal domain likely acts in concert with a fusion peptide in the S2 ectodomain to mediate final bilayer fusion once conformational changes have exposed the fusion peptide in the ectodomain. To date, there is little or no information on the fusion peptides of CoVs other than SARS-CoV, except for the identification of the N-terminal part of the mouse hepatitis virus (MHV) S HR1 domain as a putative fusion peptide based on sequence analysis (6). In none of these cases (for SARS-CoV or MHV) is the role of these sequences as bone fide fusion peptides established.The majority of class I fusion proteins prime fusion activation by proteolytic processing, with the cleavage event occurring immediately N-terminal to the fusion peptide (21). In the case of SARS-CoV, early reports analyzing heterologously expressed SARS-CoV spike protein indicated that most of the protein was not cleaved (31, 39) but that there was some possibility of limited cleavage at the S1-S2 boundary (39). However, it is generally considered that S1-S2 cleavage is not directly linked to fusion peptide exposure in the case of SARS-CoV or any other CoV (4). Recently, however, it has been shown that SARS-CoV S can be proteolytically cleaved at a downstream position in S2, at residue 797 (2, 36). Here, we investigated whether cleavage at this internal position in S2 might expose a domain with properties of a viral fusion peptide. We carried out a mutagenesis study of SARS-CoV S residues 798 to 815 using cell-cell fusion and pseudovirus assays, as well as lipid mixing and structural studies of an isolated peptide, and we show the importance of this region as a novel fusion peptide for SARS-CoV.     

Isolation and Characterization of a Prolidase from Streptococcus cremoris H61

A prolidase with a molecular weight of 43,000 was purified to homogeneity from a cell-free extract of Streptococcus cremoris H61. The optimum pH of the enzyme was in the range of 6.5 to 7.5. The hydrolyzing activity was specific for dipeptides of the X-Pro type. Kinetic constants for 4 dipeptides (Leu-Pro, Phe-Pro, Val-Pro and Ala-Pro) were estimated. Km values were not very different for these substrates, but V_max values were quite different (Leu-Pro > Phe-Pro, Val-Pro > Ala-Pro). The enzyme was activated by cobalt ion and inactivated by metal-chelating agents or with 2-mercaptoethanol.     

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum ‘Bukang’ cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.     

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H⁺­PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H⁺­PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H⁺­PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H⁺­PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.