Project PXD011671

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Summary

Title

Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803

Description

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane, unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that NdbA, which accumulates in WT thylakoid membrane particularly under LAHG conditions, has a function in redox sensing and regulation, and ultimately fine-tunes the gene expression in Synechocystis cells to meet the requirements for optimal growth under LAHG conditions.

Sample Processing Protocol

Cells were collected by centrifugation at 8000 × g for 10 min at 4 °C and washed twice with 50 mM TES-KOH buffer, pH 8.0. The cell pellet was suspended in a protein extraction buffer (0.1 % (w/v) RapiGest SF (Waters Corporation, Milford, MA) in 8 M urea solubilized in 0.1 M Tris-HCl supplemented with 200 µM PMSF) together with an equal volume of acid washed glass beads (150-212 µm, Sigma). The cells were thereafter broken using a bead beater (Mini-Bead-Beater-8, Unigenetics Instruments Pvt. Ltd., India) with six cycles of 45 s shake with 3 minutes incubation on ice. Unbroken cells and glass beads were removed by centrifugation for 5 min at 1000 x g and again for 20 min at 12000 x g at room temperature. The protein concentration was measured with the Bradford method (Bio-Rad). Proteins were reduced with 5 mM dithiotreitol (DTT, Sigma), alkylated with 10 mM iodoacetamide (IAA, Sigma) and precipitated with 1:5 v/v of 50% acetone/ 50 % ethanol o/n at -20 °C. The pellet was centrifuged at 13,200 rpm at +4 °C for 30 min and solubilized by trypsin (Sequence grade Modified, Promega, Madison, WI, USA) digestion (1:100 trypsin:protein ratio) in 50 mM Tris-HCl and 5 % (v/v) acetonitrile (ACN) buffer for 4-5 hours at +37 °C shaking. Digestion was continued for an additional 15-16 hours following a second addition of trypsin in the same ratio. The digestion was stopped by adding formic acid (FA) (Sigma) to a final concentration of 0.5–1 % (v/v) to lower the pH below 2. The digestion mixture was incubated for 30 min at +37 °C at 130 rpm and the water immiscible RapiGest SF degradation products were removed by centrifugation. To desalt the samples, solid phase extraction (SPE) of the peptide mixture was performed with Wet Sep-Pak 100 mg C18 96 well plate according to manufacturer’s protocol. The eluted peptide samples were vacuum-dried (Savant SPD1010, SpeedVac Concentrator, Thermo Scientific) and solubilized in 0,1 % (v/v) FA and 2 % (v/v) ACN and stored at -80 °C prior to MS analysis. MS data was acquired automatically by using Thermo Xcalibur 3.1 software (Thermo Fisher Scientific). A data dependent acquisition method consisted of an Orbitrap MS survey scan of mass range 300-1800 m/z followed by HCD fragmentation for 12 most intense peptide ions. The spectra were registered with a resolution of 120000 and 15000 (at m/z 200) for full scan and for fragment ions, respectively, and normalized collision energy of 27 %. The AGC (automatic gain control) was set to a maximum fill time of 100 ms and 250 ms to obtain maximum number of 3e6 and 1e5 ions for MS and MS/MS scans, respectively

Data Processing Protocol

Data files were searched for protein identification against Synechocystis database retrieved from Cyanobase (Kaneko et al., 1996) (3672 entries, 23.10.2012) using Proteome Discoverer 2.2 software (Thermo Fisher Scientific) connected to an in-house server running the Mascot 2.6.1 (Perkins et al., 1999) algorithm (Matrix Science). The precursor value was restricted to monoisotopic with a mass tolerance of ±4 ppm and fragment ion mass tolerance of ±0.02 Da. Two missed cleavages were allowed and decoy searches were performed. For the validation of the spectrum identifications, we used a Percolator algorithm (Käll et al., 2007) with relaxed false discovery rate (FDR) of 0.05. The original data and protein identification files are deposited in PRIDE Archive database (Vizcaíno et al., 2016). Quantitative analysis was done in Progenesis software with global normalization and using relative quantification of proteins with at least two peptides with no conflicts per protein.

Contact

Dorota Muth-Pawlak, University of Turku
Eva Mari-Aro, Department of Biochemistry, Molecular Plant Biology, Tykistökatu 6 A , Turku 20520, Finland ( lab head )

Submission Date

13/11/2018

Publication Date

15/03/2019

Instrument

Q Exactive HF

Modification

Carbamidomethyl
Oxidation

Quantification

Not available

Experiment Type

Shotgun proteomics

Assay count

32

Publication

    Huokko T, Muth-Pawlak D, Aro EM. Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803. Plant Cell Physiol. 2019 PubMed: 30847494

Assay

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Showing 1 - 10 of 32 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 101130 171005_OEndbA_LAH_1.mzid 2158 26349 16456 46865 0
2 101131 171005_OEndbA_LAH_2.mzid 2174 26279 16603 47396 0
3 101110 171005_OEndbA_LAH_3.mzid 2058 22619 14674 40925 0
4 101111 171005_OEndbA_LAH_4.mzid 2116 25493 15767 45480 0
5 101109 171005_OEndbA_LC_1.mzid 2043 23155 14625 41865 0
6 101101 171005_OEndbA_LC_2.mzid 2063 23482 14835 43090 0
7 101123 171005_OEndbA_LC_3.mzid 2016 22493 14292 39996 0
8 101102 171005_OEndbA_LC_4.mzid 2042 22697 14170 41930 0
9 101124 171005_WT_LAH_1.mzid 2063 24890 15656 45266 0
10 101103 171005_WT_LAH_2.mzid 2085 24945 15860 43266 0