TY - JOUR
T1 - Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae
T2 - Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation
AU - Müller, Henry
AU - Lesur, Antoine
AU - Dittmar, Gunnar
AU - Gentzel, Marc
AU - Kettner, Karina
N1 - Funding Information:
We would like to express our special thanks to Thomas M. Kriegel (Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institute of Physiological Chemistry, 01307 Dresden, Germany) for initiating the present study and for his support during its realization. We would also like to express our appreciation to Sebastian Seiferheld (Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institute of Physiological Chemistry, 01307 Dresden, Germany), who helped implement the 2D-DIGE procedure utilized in the present work and provided results of his yet unpublished investigation of TDA1 -dependent proteins by employing a SILAC approach. We thank Gerhard Rödel (Technische Universität Dresden, Institute for Genetics, 01062 Dresden, Germany) for providing lab equipment and technical support.
Funding Information:
Funding was provided by the Else Kröner-Promotionskolleg at the School of Medicine of the TU Dresden (H.M.). Open Access funding enabled and organized by Projekt DEAL. The facility Molecular Analysis—Mass Spectrometry at the CMCB was generously supported by grants of the European Regional Development Fund (ERDF/EFRE) (contract 100232736) and the German Federal Ministry of Education and Research (BMBF) (program 'Unternehmen Region', Grant 03Z2ES2) (M.G.).
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
KW - Gene Expression Regulation, Fungal
KW - Glucose/metabolism
KW - Hexokinase/genetics
KW - Phosphorylation
KW - Protein Kinases/metabolism
KW - Proteomics
KW - Saccharomyces cerevisiae/enzymology
KW - Saccharomyces cerevisiae Proteins/genetics
KW - Serine/metabolism
UR - http://www.scopus.com/inward/record.url?scp=85140830027&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/36302925
U2 - 10.1038/s41598-022-21414-x
DO - 10.1038/s41598-022-21414-x
M3 - Article
C2 - 36302925
SN - 2045-2322
VL - 12
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 18084
ER -