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Date of Award

4-1994

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Richard A. Rachubinski

Abstract

The mechanism by which subcellular organelles are assembled is one of the fundamental problems of eukaryotic cell biology and biochemistry. Proteins synthesized in the cytosol are targeted to the appropriate membrane by signals usually encrypted in some primary sequence segment. Many peroxisomal proteins are targeted by the C-terminal tripeptides which are identical to or conserved variants of Ser-Lys-Leu. Mammalian peroxisomal 3-ketoacyl-CoA thiolases are targeted by a cleavable N-terminal presequence. In this thesis the targeting of Soccharomyces cerevisiae thiolase was investigated. The N-terminal 16 amino acids of S. cerevisiae thiolase were shown to be both necessary and sufficient for peroxisomal targeting in yeast. Unlike mammalian thiolases, the native thiolase of S. cerevisiae is not detectably modified by cleavage of the targeting sequence. Several amino acid residues within the targeting region that are conserved among all thiolases were altered by mutagenesis, and three critical residues were identified- Arg4, Leu5, and Leu12. A novel approach was used to demonstrate that prior to translocation into peroxisomes, thiolase can form dimers. A targeted subunit could mediate the import of a cytosolic variant of thiolase by this means. The implications of this observation with respect to the conformation of thiolase prior to and during translocation are discussed.

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