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Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae — Spot Identifications

ABSTRACT: Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2 % and 0.1 % (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are – besides the expected Hxk2 – two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Henry Mueller

LAB HEAD: Marc Gentzel

PROVIDER: PXD037135 | Pride | 2022-10-03

REPOSITORIES: Pride

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Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

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Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae — Spot Identifications

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