Date

November 2000

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Graduate Institute of Science & Technology

Abstract

This thesis describes two positive selection schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of the observation that in P. pastoris, methanol-induced pex mutants have little or no activity for alcohol oxidase (AOX), the first enzyme in the methanol metabolic pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure to allyl alcohol selectively kills wild-type cells, resulting in an enrichment for pex mutants. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD 1), an enzyme required to metabolize the formaldehyde generated by AOX's oxidation of methanol. AOX induced fld1 cells are sensitive to high levels of methanol due to the resulting toxic accumulation of formaldehyde while fld1 pex mutants, with little or no AOX activity, are not. These selections resulted in the isolation of pex alleles in previously identified PEX genes as well as the isolation of alleles in three novel PEX groups and two groups encoding putative transcription factors. From one of those novel PEX groups, PEX14 was cloned via functional complementation. We showed that Pex14p is peripherally associated with the cytosolic side of the peroxisomal membrane and is essential for the import of peroxisomal targeting signal (PTS) 1 and PTS2 proteins but not for the targeting of peroxisomal membrane proteins. We also found that Pex14p interacts directly with Pex5p, Pex7p, Pex13p and Pex17p, all components of the peroxisomal import machinery. Finally, we discovered that Pex14p is present as a mixture of phosphorylated and unphosphorylated forms and that the phosphorylated form of Pex14p is the predominant form found in complexes with Pex13p and Pex17p. We propose a trigger model for the function of phosphorylated Pex14p in which this event is key in regulating peroxisomal matrix protein import.

Identifier

doi:10.6083/M45Q4T1H

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