Biogenesis of peroxisomes in yeast

Peroxisomes are essential metabolic compartments in eukaryotic cells, mainly involved in beta-oxidation of fatty acids. Inherited lack of peroxisomal function is correlated with human diseases like Zellweger syndrome or adrenoleukodystrophy. Proteins associated with the peroxisomal matrix or the peroxisomal membrane are synthesized in the cytoplasm, and neither the process of translocation across nor of insertion into the membrane is fully understood. The experiments described in this proposal should shed some light on the mechanism of these processes. Using the yeast Saccharomyces cerevisiae as model organism we plan to - take first steps towards the establishment of an in vitro import system, since details of molecular mechanisms can be elucidated only using externally controlled conditions. - isolate vesicles produced by the overexpression of a fusion protein containing the peroxisomal membrane anchor of the Pex3 protein and to characterize them biochemically. - find interaction partners and establish a protein linkage map around Pex15p, a peroxisomal membrane protein of as yet unknown function.

Most metabolic reactions of a cell are performed by proteins and many of these reactions take place in compartments in order to keep the best possible energetic state, to ensure optimal flux of intermediates and to separate pathways of opposite direction. There are many possible ways to determine which reaction occurs in which compartment, but nowadays technical improvements provide the opportunity to define all proteins present in a compartment at a given time. Prerequisite for such an analysis is the isolation of highly purified organelles of interest. The results from this project provide the scientific community with a novel method of purification of peroxisomes, multi-purpose compartments of high flexibility, and some preliminary data about their proteinaceous content. Differential centrifugation techniques were combined with immune-affinity methods. In the course of this project a novel affinity tag was developed and constructed for that purpose. Peroxisomal membrane proteins were labelled with this tag and employed for the isolation procedure. We could show that not all peroxisomal membrane proteins are suitable for such an approach. Proteins destined to enter a compartment carry a targeting signal, and in case of peroxisomal proteins this signal is usually located at the C-terminal end of the amino acid sequence. In many cases sequence analytic tools and bioinformatics techniques are helpful in determining the protein`s structure or localization. To this end we developed together with the group of F. Eisenhaber (IMP, Vienna) an extremely reliable sequence analytic tool to predict the peroxisomal location of a given protein.