Constitutive Biosynthesis and Localization of Alcohol Oxidase in the Ethanol-Insensitive Catabolite Repression Mutant ecr1 of the Yeast Pichia methanolica

K. A. Lusta¹, O. A. Leonovitch², I. I. Tolstorukov³, and Ya. M. Rabinovich^2*

¹Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, 142292 Russia; fax: (7-095) 923-3602; E-mail: lusta@ibpm.serpukhov.su

²Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax (7-095) 954-2732; E-mail: inbio@glas.apc.org

³State Scientific Research Institute of Genetics and Selection of Technical Microorganisms, 1-yi Dorozhnyi Proezd 1, Moscow, 113545 Russia; fax: (7-095) 315-0501; E-mail: ilya@vnigen.msk.su

^* To whom correspondence should be addressed.

Received July 12, 1999; Revision received October 25, 1999

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The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm³) during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.

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KEY WORDS: alcohol oxidase, biogenesis of peroxisomes, methylotrophic yeast, Pichia methanolica, catabolite repression

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