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Journal of Bacteriology, December 2000, p. 7007-7013, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

Marijke A. H. Luttik,1 Peter Kötter,2 Florian A. Salomons,3 Ida J. van der Klei,3 Johannes P. van Dijken,1 and Jack T. Pronk1,*

Kluyver Laboratory of Biotechnology, Department of Microbiology and Enzymology, Delft University of Technology, 2628 BC Delft,1 and Biological Centre, Department of Microbiology, University of Groningen, 9751 NN Haren,3 The Netherlands, and Institut für Mikrobiologie, J. W. Goethe Universität Frankfurt, Biozentrum N250, 60439 Frankfurt, Germany2

Received 17 May 2000/Accepted 27 September 2000

The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect of icl1 null mutants. It has therefore been suggested that ICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whether ICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenic icl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 and icl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2 mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that the ICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction of ICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

* Corresponding author. Mailing address: Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 278 3214. Fax: 31 15 278 2355. E-mail: j.t.pronk{at}stm.tudelft.nl.

Journal of Bacteriology, December 2000, p. 7007-7013, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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