An efficient screen for peroxisome-deficient mutants of Pichia pastoris

HongYan Liu, X. Tan, M. Veenhuis, D. McCollum, J. M. Cregg

Research output: Contribution to journalArticle

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Abstract

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.

Original languageEnglish (US)
Pages (from-to)4943-4951
Number of pages9
JournalJournal of Bacteriology
Volume174
Issue number15
DOIs
StatePublished - Jan 1 1992

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Peroxisomes
Pichia
Methanol
Oleic Acid
alcohol oxidase
Enzymes
Cytosol
Catalase
Molecular Biology
Electron Microscopy
Ethanol
Carbon
Yeasts
Phenotype
Glucose
Mutation
Growth

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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An efficient screen for peroxisome-deficient mutants of Pichia pastoris. / Liu, HongYan; Tan, X.; Veenhuis, M.; McCollum, D.; Cregg, J. M.

In: Journal of Bacteriology, Vol. 174, No. 15, 01.01.1992, p. 4943-4951.

Research output: Contribution to journalArticle

Liu, H, Tan, X, Veenhuis, M, McCollum, D & Cregg, JM 1992, 'An efficient screen for peroxisome-deficient mutants of Pichia pastoris', Journal of Bacteriology, vol. 174, no. 15, pp. 4943-4951. https://doi.org/10.1128/jb.174.15.4943-4951.1992
Liu, HongYan ; Tan, X. ; Veenhuis, M. ; McCollum, D. ; Cregg, J. M. / An efficient screen for peroxisome-deficient mutants of Pichia pastoris. In: Journal of Bacteriology. 1992 ; Vol. 174, No. 15. pp. 4943-4951.
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