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Date of Award

4-1992

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Richard A. Rachubinski

Abstract

The nature of the peroxisomal targeting signal in Candida tropicalis trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE) was investigated. The first part of this thesis describes the cloning and sequencing of the cDNA and gene encoding HDE. The second and third parts of the thesis describe the amino acid sequence (and general nature) of the signal that directs HDE to peroxisomes in yeast. The cDNA and gene encoding C. tropicalis HDE was isolated from a λgt11 cDNA expression library and from a λEMBL3 genomic library, respectively. Primary sequence analysis of both the cDNA and the gene revealed a single open reading frame of 2718 nucleotides encoding a protein of 906 amino acids (calculated molecular mass = 99,481 Da). The gene encoding HDE was expressed in the yeasts Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxy-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. Substitutions within the carboxy-terminal tripeptide (Ala → Gly and Lys → Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. Antiserum directed against the carboxy-terminal 11 amino acids of HDE, containing the Ala-Lys-Ile tripeptide (anti-AKI) was used to probe subcellular fractions from several yeasts and rat liver. The anti-AKI serum reacted exclusively with multiple peroxisomal proteins from the yeasts C. tropicalis, C. albicans and Yarrowia lipolytica suggesting a common motif for the targeting of some proteins to yeast peroxisomes, the prototype of which is the HDE carboxy-terminal tripeptide Ala-Lys-Ile.

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