Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

ATPG is required for the accumulation and function of chloroplast ATP synthase in Arabidopsis

The subunit Ⅱ of chloroplast ATP synthase is one of the two peripheral stalks, which associates the catalytic CF1 with mem-brane-spanning CFo . Although the structural and functional roles of chloroplast ATP synthase have been extensively examined, the physiological significance of subunit Ⅱ in vivo is still unclear. In this work, we identified one Arabidopsis T-DNA insertion mutant of atpG gene encoding the subunit Ⅱ of chloroplast ATP synthase. The atpg null mutant displayed an albino lethal pheno-type, as it could not grow photoautotrophically. Transmission electron microscopy analysis showed that chloroplasts of atpg lacked the organized thylakoid membranes. Loss of subunit Ⅱ affected the accumulation of CF1-CFo complex, however, it did not seem to have an effect on the CF1 assembly. The light induced ATP formation of atpg was significantly reduced compared with the wild type. Based on these results, we suggested that ATPG was essential for the accumulation and function of chloroplast ATP synthase.     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

The short C-terminal hydrophilic domain of NhaH Na~ /H~ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na /H antiporter plays key roles in maintaining low cytoplasmic Na level and pH homeostasis,while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters.In our previous study,the first Na /H antiporter gene nhaH from moderate halophiles was cloned from Halo-bacillus dabanensis D-8 by functional complementation.A topological model suggested that only nine amino acid residues(395PLIKKLGMI403) existed in the hydrophilic C-terminal domain of NhaH.The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaH△C.Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid resi-dues significantly inhibited the complementation ability of E.coli KNabc,in which three main Na /H antiporters nhaA,nhaB and chaA were deleted.Everted membrane vesicles prepared from E.coli KNabc/nhaH△C decreased both Na /H and Li /H exchange activities of NhaH,and also resulted in an acidic shift of its pH profile for Na ,indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.     

The short C-terminal hydrophilic domain of NhaH Na^＋/H^＋ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na^＋/H^＋ antiporter plays key roles in maintaining low cytoplasmic NaNa^＋ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na^＋/H^＋ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues （^395PLIKKLGMI403） existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaH△C. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coil KNabc, in which three main Na^＋/H^＋ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coil KNabc/nhaHAC decreased both Na^＋/H^＋ and Li^＋/H^＋ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na^＋, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.     

Localization of NOS-like protein in guard cells of Vicia faba L. and its possible function

Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of ni- tric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS in- hibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO genera- tion from NOS pathway is the main pathway in the stress and JA responses. The pharmacological ex- periment showed that increasing the Ca2 at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2 -dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.     

Nucleotide sequence of RNA2 and polyprotein processing sites of a Chinese isolate of broad bean wilt virus 2

RNA2 of broad bean wilt virus 2 (BBWV2) isolate B935 is composed of 3601 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six possible reading frames has a long open reading frame, which extends from nt 231 to nt 3428 in the polarity of encapsidated RNA, and encodes a polyprotein of 119 kD. The N-terminus of the large coat protein (LCP) is located at 599 nt and the small coat protein (SCP) at 197 nl from the C-terminus of the 119 kD protein, which suggests that the coat proteins are released from the polyprotein by cleavages of a glulamine-glycine (Q-G) and a glutamine-alanine (Q-A) bond respectively. The sequence comparison of B935 with fabaviruses shows that B935 has very high sequence homology with other BBWV2 isolates and with patchoul' mild mosaic virus, but has lower homology with BBWV1 isolates. B935 has a similar genomic organization, but a low sequence homology to RNA2 molecules of comoviruses and nepoviruses.     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-l in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2 . On the basis of the research on ftsZl family, we analyzed the function of NtFtsZ2-l gene in Nicotiana tabacum . Microscopic analysis of the sense and antisense NtFtsZ2-l transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-l gene is involved in plant chloroplast division.     

Distribution of lanthanum among the chloroplast subcomponents of spinach and its biological effects on photosynthesis： location of the lanthanum binding sites in photosystem Ⅱ

The effects of lanthanum at different concentrations on the related photosynthetic activities of Hill reaction, Mg^2＋-ATPase and Ca^2＋-ATPase in spinach chloroplast were studied. Experimental results showed that lanthanum can increase all the activities at suitable concentration （15-30 mg· L^-1）, however, it behaves toxically on them when over used （60 mg. L^-1）. To get an improved understanding of the mechanism of lanthanum effects on the photosynthesis of spinach, the different subcomponents in the chloroplast of the cultured spinach were isolated, and the content of lanthanum in each subcomponent was determined by ICP-MS. The results obtained indicated that among these different subcomponents, about 90% out of the total chloroplast lanthanum was located in photosystem Ⅱ （PS Ⅱ） while there was little lanthanum in photosystem Ⅰ （PS Ⅰ）. Moreover, size exclusion high performance liquid chromatography （SE-HPLC） coupled with online UV and ICP-MS detections was novelly used for locating lanthanum binding sites in PS Ⅱ proteins for the first time. It was found that lanthanum has two binding sites in PS Ⅱ： La associates with chlorophyll together with magnesium in PS Ⅱ by partly replacing magnesium and also shares the common binding sites of PS Ⅱ proteins together with the inorganic cofactors of calcium and manganese, influencing the process of photosynthesis.     

Expression，reflolding and preliminary characterization of recombinant snake venom metalloproteinases：Implication for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase(BR)were cloned into the expression vector pET-22b,respectively,and the corresponding two recombinant proteins,A-22b and BR-22b,were produced in inclusion bodies in E.coli BL21(DE3).The reombinant proteins were then subjected to solubilization,purification and refolding in vitro.A-22b showed hemorrhagic activity and fibronectin.Natural autolysin A had both hemorrhagic activity and proteolytic activity toward these substrates.BR-22b showed the proteolytic activities toward fibrinogen,but no hemorrhagic activity.In addition,two chimeric genes,C1 and C2,were constructed and cloned into pET-22b,and the corresponding recombinant proteins,C1-22b and C2-22b,were also expressed in inclusion bodies.C1-22b involved N-terminal 110 amino acidos of BR and C-terminal 95 amino acids of acutolysin A,while C2-22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of A-22b and BR-22b,respectively.Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.     

Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-1 in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2. On the basis of the research on ftsZ1 family, we analyzed the function of NtFtsZ2-1 gene in Nicotiana tabacum. Microscopic analysis of the sense and antisense NtFtsZ2-1 transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-1 gene is involved in plant chloroplast division.     

Dithiothreitol decreases the thermal stability and unfolding cooperativity of ribulose-1, 5-bisphosphate carboxylase/oxygenase

Plant rubisco consists of eight large subunits (55 kD) encoded by chloroplast gene and eight small subunits (15 kD) encoded by nuclear gene. There are abundant cysteine residues that do not form disulfide bonds in native rubisco. Differential scanning calorimetry has been used to study some plant rubisco and suggested an irreversible two-state denaturation due to the high cooperativity in subunits. By comparing the data from circular dichroism, fluorescence, differential scanning calorimetry, SDS electrophoresis, and activity assays in the absence or presence of DTT, we suggest that the formation of disulfide bonds in subunits during the early thermal unfolding may increase the thermal stability and the thermal unfolding cooperativity of rubisco.     

Synthesis of medium-chain-length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering

Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3‘-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line $4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mclPHAs in tobacco via chloroplast genetic engineering.     

Study on the interaction between Jak3 and IL-2R γ using the yeast two-hybrid system

Both interleukin-2 (IL-2) receptor γ subunit and non-receptor tyrosine kinase Jak3 play important roles in IL-2 physiological functions. Jak3 has been known to bind IL-2Rγ and the interaction is very important for IL-2 signaling. In order to find the domains directly involved in the interaction between IL-2Rγ and Jak3, various deletion mutants have been constructed and their interaction has been studied using the yeast two-hybrid system. Results show that the JH3-JH7 region of Jak3 N-terminal can bind to intracellular domain of IL-2Ry directly and the intact structure of JH3-JH7 region is necessary for this combination. In addition, the region from 305 to 317 amino acid residues near the C-terminal of IL-2Ry plays a critical role in this interaction .