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Date of Award

6-1991

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

R.A. Rachubinski

Abstract

This thesis is divided into two parts, each part addressing a particular aspect of the biogenesis of rat liver peroxisomes. The first part describes the characterization of the peroxisomal integral membrane polypeptides, and the second part describes the primary structure and targeting of peroxisomal 3-ketoacyl-CoA thiolase.

In the first part, membranes were isolated from liver peroxisomes of untreated rats and rats treated with clofibrate, a hypolipidemic drug which induces peroxisomal proliferation in rodent hepatocytes. The membranes were used to raise an antiserum in rabbits that reacted with six peroxisomal integral membrane polypeptides (molecular masses 140, 69, 50, 36, 22 and 15 kDa). Treatment of rats with clofibrate caused a 4- to 10-fold induction in the 69 kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50 kDa and 36 kDa integral membrane polypeptides of peroxisomes. Subcellular fractionations and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22, 36, and 69 kDa integral membrane polypeptides were synthesized on free polysomes, while the 50 kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50 kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis and trafficking.

3-Ketoacyl-CoA thiolase (thiolase) catalyzes the final step of the fatty acid a-oxidation pathway in peroxisomes. Thiolase is unique among rat liver peroxisomal enzymes in that it is synthesized as a precursor possessing an amino-terminal extension, which is cleaved to generate the mature enzyme. To facilitate further examination of the synthesis, intracellular transport and processing of this enzyme, cDNA recombinants encoding thiolase were selected from a λgt11 rat liver library using antiserum raised against peroxisomal thiolase. Upon sequencing several cDNA recombinants, it was revealed that there are two distinct thiolase enzymes in rat liver peroxisomes, thiolase 1 and thiolase 2. The cDNAs encoding thiolases 1 and 2 exhibited 94.6% nucleotide sequence identity and 95.4% identity at the level of deduced amino acid sequence. Interestingly, the expression of the genes encoding the two thiolases was differentially regulated. The mRNA encoding thiolase 1 was expressed at a low level in untreated rat liver and was induced greater than 10-fold upon treatment with clofibrate. In contrast, the mRNA encoding thiolase 2 appeared to be constitutively expressed in rat liver and was only slightly induced upon treatment of rats with clofibrate. The cDNA encoding peroxisomal thiolase 2 was used in in vivo targeting studies which indicated that the thiolase prepiece contains information for targeting this protein to peroxisomes.

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