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Regulation of the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces

T J Wenzel1, M A Luttik, J A van den Berg

  • 1Department of Molecular and Cellular Biology, Leiden University, The Netherlands.

European Journal of Biochemistry
|December 1, 1993
PubMed

Insights

Pyruvate dehydrogenase complex (PDH complex) expression is constitutive. PDH complex activity, not gene expression, is regulated post-translationally, suggesting roles beyond sugar metabolism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Metabolism

Background:

  • The pyruvate dehydrogenase complex (PDH complex) is crucial for cellular energy production.
  • Understanding the regulation of PDH complex gene expression and activity is key to comprehending metabolic control.

Purpose of the Study:

  • To investigate the regulation of PDA1 gene expression and PDH complex activity under various growth conditions.
  • To determine the role of the GCN4 mechanism in regulating PDA1 expression.
  • To elucidate the primary regulatory mechanisms controlling PDH complex activity.

Main Methods:

  • Analysis of PDA1 mRNA levels using techniques like Northern blotting or RT-qPCR.
  • Quantification of E1 alpha subunit protein levels via Western blotting or ELISA.
  • Measurement of PDH complex activity using spectrophotometric assays.
  • Cell cultivation under different conditions, including batch and chemostat cultures, and varying carbon sources.
  • Manipulation of GCN4 pathway using 3-amino-1,2,4-triazole.
  • Overproduction of the E1 alpha subunit.

Main Results:

  • PDA1 mRNA and E1 alpha subunit levels were largely constant across different carbon sources and culture types, indicating constitutive expression.
  • Histidine starvation induced by 3-amino-1,2,4-triazole increased PDA1 mRNA by ~40% but did not affect E1 alpha levels or PDH complex activity.
  • PDH complex activity was significantly higher (2-3 fold) in chemostat cultures compared to batch cultures.
  • Overproduction of the E1 alpha subunit in batch cultures also led to a 2-3 fold increase in PDH complex activity.
  • PDH complex activity and PDA1 expression were detected even in conditions where its role in sugar catabolism is minimal (anaerobic growth on glucose, aerobic growth on ethanol).

Conclusions:

  • PDH complex gene expression (PDA1) is primarily constitutive and minimally regulated by the GCN4 mechanism.
  • PDH complex activity is mainly controlled by post-translational modifications of the E1 alpha subunit, rather than transcriptional or translational regulation.
  • The PDH complex may possess functions beyond its established role in carbohydrate metabolism, as indicated by its activity under non-standard growth conditions.

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