Characterization of Saccharomyces cerevisiae mutants lacking the E1α subunit of the pyruvate dehydrogenase complex

Authors


Correspondence to T. J. Wenzel, Clusius Laboratorium, Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands
Fax: +31 71 27 48 63.

Abstract

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDAl gene. To this end, the PDAl gene encoding the E1α subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDAl gene abolished production of the E1α subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho°], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDAl gene. Explanations for these phenotypes are discussed.

Abbreviations
PCR

polymerase chain reaction

Nbs2

5,5′-dithiobis(2-nitrobenzoic acid)

G418

Geneticin

dig-dUTP

Digoxigenin-11-dUTP

Enzymes
 

2-Isopropylmalate synthase (EC 4.1.3.12)

 

dihydrolipoamide dehydrogenase (EC 1.8.1.4)

 

lipoate acetyltransferase (EC 2.3.1.12)

 

pyruvate decarboxylase (EC 4.1.1.1)

 

pyruvate dehydrogenase (EC 1.2.4.1)

 

restriction endonucleases (EC 3.1.21.4)

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