Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.     

Aerobic metabolism of phenylacetic acids in Azoarcus evansii

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.     

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.     

PR65A 调控抗病毒蛋白ZAP的活性

锌指结构抗病毒蛋白(ZAP)能够特异性地识别病毒RNA并促进其特异性降解,从而抑制病毒的复制．ZAP不能独立地发挥抗病毒的功能,需要招募细胞内很多因子,包括外切酶复合体(exosome)、RNA解旋酶p72 等．通过蛋白质组学分析方法,我们找到了与ZAP可能存在相互作用的一些蛋白质并证实蛋白磷酸酶2调节亚基A的?琢亚型(PR65A)与ZAP之间存在着直接的相互作用．细胞内PR65A的表达水平,降低削弱ZAP的活性, 表明PR65A是ZAP发挥最佳抗病毒活性所必需的．这丰富了我们对ZAP抗病毒作用机理的认识,同时也为更好地利用其抗病毒活性提供了重要的指导．     

alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure

Protein phosphatase 2A (polycation-stimulated protein phosphatase L) was purified from porcine kidney and skeletal muscle. The 36-kDa catalytic and the 65-kDa putative regulatory (hereafter termed PR65) subunits of protein phosphatase 2A2 were separated by reverse-phase HPLC. Partial amino acid sequence data (300 residues) was obtained for PR65. Molecular cloning showed that two distinct mRNAs (termed alpha and beta) encoded the PR65 subunit. The cDNA encoding the alpha-isotype spanned 2.2 kilobases (kb) and contained an open reading frame of 1767 bases predicting a protein of 65 kDa, which was in good agreement with the size of the purified protein. The cDNAs encoding the beta-isotype contained an open reading frame of size similar to that of alpha-form but lacked an initiator ATG. Northern analysis, using RNA isolated from several human cell lines, indicated that the alpha-isotype was encoded by a mRNA of 2.4 kb that was much more abundant than the beta mRNA of 4.0 kb. Comparison of the predicted amino acid sequences of the two isotypes revealed 87% identity. The deduced protein sequences of the alpha- and beta-isotypes were found to be made up of 15 imperfect repeating units consisting of 39 amino acids. This repeating structure was conserved between species.     

The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation

Physiological functions of protein phosphatase 2A (PP2A) are determined via the association of its catalytic subunit (PP2Ac) with diverse regulatory subunits. The predominant form of PP2Ac assembles into a heterotrimer comprising the scaffolding PR65/A subunit together with a variable regulatory B subunit. A distinct population of PP2Ac associates with the Tap42/alpha4 subunit, an interaction mutually exclusive with that of PR65/A. Tap42/alpha4 is also an interacting subunit of the PP2Ac-related phosphatases, PP4 and PP6. Tap42/alpha4, an essential protein in yeast and suppressor of apoptosis in mammals, contributes to critical cellular functions including the Tor signaling pathway. Here, we describe the crystal structure of the PP2Ac-interaction domain of Saccharomyces cerevisiae Tap42. The structure reveals an all alpha-helical protein with striking similarity to 14-3-3 and tetratricopeptide repeat (TPR) proteins. Mutational analyses of structurally conserved regions of Tap42/alpha4 identified a positively charged region critical for its interactions with PP2Ac. We propose a scaffolding function for Tap42/alpha4 whereby the interaction of PP2Ac at its N-terminus promotes the dephosphorylation of substrates recruited to the C-terminal region of the molecule.     

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.     

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation.     

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.     

A new pathogenesis-related protein,LrPR4, from <Emphasis Type="Italic">Lycoris radiata</Emphasis>, and its antifungal activity against <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A new Lycoris radiata pathogenesis-related (PR)-4 gene, LrPR4 was isolated. LrPR4 encodes a 142 amino acid protein with a predicted molecular mass of 15.43 kDa and pI of 7.56. The putative LrPR4 shows high similarity to PR4 type proteins from various plant species and belongs to the Barwin family. Like other PR4s from monocot plants, LrPR4 protein contains a conserved Barwin domain and has a signal peptide at its N-terminus. The recombinant LrPR4 protein expressed in Escherichia coli showed activity towards hydrolysing RNA from L. radiata bulbs and antifungal activity. The results of this study suggest that LrPR4 may play a role in the disease resistance responses of plant against pathogen attacks though its antifungal activity.     

Isolation and characterization of a cDNA encoding a synaptonemal complex protein.

A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis lambda gt11 and lambda ZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50,000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10(5)-1.5 x 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases.