Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting

Simon, William J.; Hall, John J.; Suzuki, Iwane; Murata, Norio; Slabas, Antoni R.

doi:10.1002/1615-9861(200212)2:12<1735::aid-prot1735>3.0.co;2-k

Cited by 43 publications

(19 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Crucially, FutA1 has never been observed in soluble periplasmic extracts (40,43). Conversely, as here, FutA2 was previously found in periplasm extracts released by cold osmotic shock (40), although it has also been detected in preparations of plasma membranes (41), thylakoid membranes (43,44), and total soluble extracts (43,45), the latter possibly including a subpopulation of folded FutA2 awaiting export via the TAT pathway. Cells deficient in FutA2 also accumulate a low M r pigmented compound at the cell surface (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

Waldron¹,

Tottey²,

Yanagisawa

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c 6 when grown under copper conditions (150 nM) in which wild-type cells use plastocyanin rather than cytochrome c 6 . Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copperplastocyanin is impaired, but iron-ferredoxin is unaffected in ⌬futA2 grown in 150 nM copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in ⌬futA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of ⌬futA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in ⌬futA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.Oxygenic phototrophs have exceptional requirements for metals such as iron, copper, and manganese within the photosynthetic machinery (1, 2). There is interest in understanding how metal ions partition to the correct cellular destinations in all organisms (3), including to the approximately one-third of proteins that need metals (4). The periplasm is a key location for metal partitioning. Bacterial ATP binding cassette (ABC)-type importers include substrate binding proteins which may be free within the periplasm or anchored to the outer face of the plasma membrane. It is anticipated that these proteins assist in loading metal ions onto the correct importers. There is similarity in the metal-binding sites of several substrate binding proteins that contribute toward the import of distinct metals, such as manganese versus zinc, and subtle differences in second coordination spheres may be crucial for metal selectivity (5). It has also been noted that the nature of interactions between substrate binding proteins and their respective metal transporters may make a key contribution to metal specificity (5

show abstract

Section: Discussionmentioning

confidence: 99%

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

Waldron¹,

Tottey²,

Yanagisawa

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The second and third columns show changes in relative levels of proteins after heat shock due to incubation at 42°C for 60 min. This table lists six proteins that we identified in previous studies (Simon et al, 2002) and that we also identified as products of heat-inducible genes by DNA microarray analysis (Table III). The values are averages of results of five independent experiments, which gave a statistical confidence limit of 95%.…”

Section: A Possible Scheme For Regulation Of Gene Expression By Hik34mentioning

confidence: 98%

“…In a previous study of proteins in Synechocystis, we separated more than 300 protein spots and identified more than 100 soluble proteins from Synechocystis cells by two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry (Simon et al, 2002). These proteins included HSPs such as GroEL, DnaK2, HspA, and ClpB.…”

Section: Analysis Of Hsps In Wild-type and Dhik34 Cellsmentioning

confidence: 99%

See 1 more Smart Citation

The Histidine Kinase Hik34 Is Involved in Thermotolerance by Regulating the Expression of Heat Shock Genes in Synechocystis

et al. 2005

Self Cite

View full text Add to dashboard Cite

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (DHik34 cells) survived incubation at 48°C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44°C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in DHik34 cells after incubation of cells at 42°C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44°C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

show abstract

“…Tryptic digests from each peptides were separated with a MALDI TOF spectrophotometer (ABI Voyager DE-STR) as described in Simon et al (2002). Similarly the tryptic digest from the purified protein associated with UDPGDH and ADH activities was analysed with a MALDI-TOF (Brucker Reflex 3).…”

Section: Maldi Tof Analysis Of Purified Proteinsmentioning

confidence: 99%

Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue

Bindschedler

Wheatley

Cole³

et al. 2005

Plant Mol Biol

View full text Add to dashboard Cite

The pathway from UDP-glucose to UDP-xylose has been characterised in differentiating tobacco tissue. A xylogenic suspension cell culture of tobacco has been used as a source for the purification of the enzymes responsible for the oxidation of UDP-glucose to UDP-glucuronic acid and its subsequent decarboxylation to UDP-xylose. Protein purification and transcriptional studies show that two possible candidates can contribute to the first reaction. Most of the enzyme activity in the cultured cells could be accounted for by a protein with an Mr of 43 kDa which had dual specificity for UDP-glucose and ethanol. The cognate cDNA, with similarity to alcohol dehydrogenases (NtADH2) was expressed in E. coli to confirm the dual specificity. A second UDP-glucose dehydrogenase, corresponding to the monospecific form, ubiquitous amongst plants and animals, could not be purified from the tobacco cell cultures. However, two cDNAs were cloned with high similarity to the family of UDP-glucose dehydrogenases. Transcripts of both types of dehydrogenase showed highest expression in tissues undergoing secondary wall synthesis. The UDP-glucuronate decarboxylase was purified as polypeptides of Mr 87 and 40 kDa. Peptide fingerprinting of the latter polypeptide identified it as a form of UDP-glucuronate decarboxylase and functionality was established by expressing the cognate cDNA in E. coli. Expression of 40 kDa polypeptide and its corresponding mRNA was also found to be highest in tissues associated with secondary wall formation.

show abstract

Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting

Cited by 43 publications

References 16 publications

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

The Histidine Kinase Hik34 Is Involved in Thermotolerance by Regulating the Expression of Heat Shock Genes in Synechocystis

Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue

Contact Info

Product

Resources

About