Date of Award

3-1992

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

R.A. Rachubinski

Abstract

Studies concerned with peroximes biogenesis have established that peroxisomal proteins are encoded exclusively by nuclear genes, synthesized (with few exceptions) on free polyribosomes at their mature size and posttranslationally imported into preexisting oganelles. From this, it was realized that the information responsible for the specificity of protein targeting to peroxisomes must reside within the mature amino acid sequence of the imported protein. Knowledge of the targeting sequences of peroxisomal proteins is, however, limited. Consequently, using the yeast C. tropicalis pK233 as a model system, this work endeavoured to clone and sequence a cDNA or gene encoding a peroxisomal protein so that the subsequent creation of deletion mutants of this cDNA/gene, together with the development of and in vitro and/or in vivo expression and translocation system for the corresponding polypeptides, would allow the amino acid sequence(s) comprising the peroxisomal addressing signal of this protein to be identified.

Related efforts resulted in the cloning of two genes (and one composite full-length cDNA) encoding different enzymes associated with the C. tropicalis peroxisomes. Sequencing of the gene encoding acyl-coenzymes A oxidase (AOx), revealed a single open reading frame, without intervening sequences, of 2,127 nucleotides which encodes a polypeptide of 709 amino acids (Mr 79155). The deduced amino acid sequence of C. tropicalis AOx exhibits significant identity and homology with that of several other acyl-coenzyme A oxidases of C. tropicalis, as well as other yeasts (C. maltosa and S, cerevisiae) and rat. By comparison, sequencing of the C. tropicalis gene (or cDNA) encoding catalase (Cat) revealed a single open reading frame of 1,455 nucleotides, without intervening sequences, which encodes a polpeptide of 485 amino acid (Mr 54944). The deduced amino acid equence of C. tropicalis Cat shows sigificant identity and homology with that of catalases from the yeast S. cerevisiae mammals (rat, human and bovine), plants (maize, sweet potato and cottonseed) and Drosophila. COdon usage in the genes encoding C.tropicalis AOx and Cat is nonrandom, exhibiting a strong bias towards a group of preferred codons of S. cerevisiae and E. coli, as weel as other C. tropicalis genes encoding peroxisomal proteins.

Having achieved the charaterization of the genes encoding C. tropocalis AOx and Cat, attempts were made to establish an in vitro system for the translocation of Cat into peroxisomes purified from C. tropicalis which could be used to determine the peroxisomal targeting signal (PTS) of Cat. For this purpose, N-terminal, C-terminal, N-/C-terminal and internal deletion mutants of Cat were constructed, expressed in cell-free systems and assayed for translocation into purified C. tropicalis perxisomes. Unfortunately, all attempts at in vitro imports proved to be inadequate for the translocation of Cat into purified C. tropicalis peroxisomes. Since a functional in vitro system could not be established, studies concerned with the in vivo expression and targeting of Cat from C. tropicalis to the peroxisomes of C. albicans and S. cerevisiae were initiated. Although, the in vivo expression of Cat sequences could not be attained in C. albicans, the expression of full-length and truncated Cat polypeptides was achieved in S. cerevisiae. Subsequent determination of the subcellular location of the full-length Cat polypeptide suggested that Cat from C. tropicalis was targeted to the peroxisomes of S. cerevisiae, reaffirming that heterologous signals for import into peroxisomes are recognized in S. cerevisiae. Interestingly, the deletion of amino acids 182 to 344 of Cat negated the targeting of Cat to S. cerevisiae peroxisomes. These preliminary in vivo studies suggest that at least part of the PTS of Cat from C. tropicalis resides at an internal location within its mature amino acid sequence.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS