Peroxisomes are nearly ubiquitous in eukaryotic cells and have essential functions in respiration and lipid metabolism. Peroxisomal proteins are nuclear-encoded and are synthesized on free polyribosomes, then imported into pre-existing peroxisomes post-translationally. Peroxisomal proteins, unlike many mitochondrial and chloroplast proteins, are synthesized at their mature size and lack cleavable transit peptides. The long term objective of this project is to understand the mechanism of peroxisome assembly. The aims of this proposal are to identify topogenic sequences that act to direct peroxisomal proteins to yeast peroxisomes. Previously, this laboratory determined conditions for measuring the in vitro import of peroxisomal proteins to peroxisomes from the yeast Candida tropicalis, and identified topogenic sequences in one peroxosomal protein (aceyl-CoA oxidase) that are capable of direction this protein to peroxisomes. Nothing is known regarding the targeting and insertion of peroxisomal integral membrane proteins. The plan is to clone and sequence a matrix and membrane protein from C. tropicalis peroxisomes. Using the established in vitro assay and by an in vivo import assay, peroxisomal targetting signals will be investigated and compared. Also, the gene encloding peroxisomal citrate synthase from Saccharomyces cerevisiae is in hand; this will be expressed and, using the above methods, it will be determined whether signal sequences are conserved in peroxisomal proteins from C. tropicalis and S. cerevisiae. By these means, it is hoped to gain a better understanding of the mechanism by which peroxisomes are assembled.
