9514289 Cregg Peroxisomes are present in the cells of virtually all eukaryotes and are the subcellular location of important metabolic reactions. Yet relatively little is known about the basic mechanisms by which cells maintain and propagate peroxisomes and direct specific sets of newly synthesized proteins to these organelles (peroxisome biogenesis). Recent studies indicate that peroxisome biogenesis mechanisms are novel and not simply variations of those in other organelles. A molecular genetic investigation of peroxisomes has been initiated using a methanol-utilizing yeast, Pichia pastoris, as a model system. This yeast was selected because: (1) peroxlsomes are required for its growth on either methanol or oleic acid, phenotypes that are easily observed, and (2) the organelles are large and numerous in cells grown on these substrates. In the previous grant period, a number of peroxisome-deficient (per) mutants of P. pastoris were isolated, and significant progress was made in the cloning and characterization of several of the affected PER genes. This proposal is aimed at elucidating the structure and function of two very interesting P. pastoris PER gene products, Per3p and Per6p. Per3p is an 81-kDa membrane-associated protein that appears to be located on the inner surface of the peroxisomal membrane. Per3p is involved in import of peroxisomal matrix enzymes and may be composed of multiple domains required for the function of different peroxisomal import pathways. To test this multi-domain hypothesis and to define the approximate location of the domains, mutations will be introduced into PER3, and their effects on the function of specific import pathways will be determined. Per3p is one of only a few peroxisomal import machinery components that has been identified. To identify other import components, the two-hybrid protein method will be used to search for proteins that interact with Per3p. These studies will provide valuable insights into the role of P er3p in protein import and into the identity and organization of other components of the import machinery. Per6p is a 52-kDa peroxisomal integral membrane protein that has similarity to PAFlp, a protein affected in certain patients with the peroxisomal biogenesis disorder Zellweger syndrome. Although it is suspected that these proteins are involved in peroxisomal protein import, direct evidence for this is lacking. To obtain such evidence for Per6p, temperature-sensitive per6 strains will be shifted to nonpermissive temperature, and the fate of import of newly synthesized (pulse-labeled) peroxisomal proteins will be examined. If Per6p is required for the import, import should rapidly cease after shift. Peroxisomal targeting signals (PTSs) have been identified for matrix proteins but not for peroxisomal integral membrane proteins. To identify PTS sequences in Per6p, selected NH2- and COOH-terminally truncated derivatives of the protein will be synthesized in P. pastoris and examined for proper targeting. Defining a PTS for membrane proteins is a critical first step in understanding how cells target and insert this class of proteins into the peroxisomal membrane. ***
