Peroxisomes are ubiquitous eukaryotic organelles involved in a variety of important metabolic pathways. In humans, peroxisomes are essential for the metabolism of certain lipids, and defects in their biogenesis are responsible for a family of lethal genetic disorders, collectively termed Zellweger syndrome. The primary long-term goal of this program is to understand, at the molecular level, the mechanisms controlling peroxisomal biogenesis. A combined molecular genetic and biochemical attack has been initiated using the years Pichia pastoris as the model system. In recent years, more than a dozen different PEX genes and their products have been described. However, convincing evidence for a specific role in biogenesis has been provided for only a few of these. A major goal of this proposal is to establish the specific function of two P. pastoris PEX proteins, Pex2p and Pex8p, in peroxisome biogenesis. Pex8p is a protein located on the inner membrane of the peroxisome that may function to release newly imported matrix proteins from their receptors. Evidence for this model will be obtained by identifying and dissecting domains on Pex8p responsible for import of specific subsets of peroxisomal proteins and for interaction with specific import receptors. Pex2p belongs to a family of related peroxisomal integral membrane proteins and is the P. pastoris ortholog of a Zellweger gene. Members of this family are hypothesized to form the core of the peroxisomal protein translocation apparatus. Pulse-chase studies will be conducted on temperature-sensitive pex2 mutants to obtain evidence that Pex2p function is required for protein import. The organization of the putative import apparatus will be elucidated through the identification of Pex2p interacting proteins. A combination of approaches involving the two-hybrid system, suppressor mutant analysis and purification of Pex2p-containing complexes will be employed. Finally, a highly efficient selection scheme will be utilized to identify P. pastoris mutants defective in novel PEX genes. The PEX genes will be cloned, and the primary sequences of their products will be used to search the databases for their human PEX cDNA orthologs. Human PEX cDNAs that are also Zellweger genes will be identified by their ability to restore normal peroxisomes to specific Zellweger cell lines. Thus, this yeast system provides an excellent model for investigating peroxisome biogenesis and understanding the molecular etiology of this disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043698-08
Application #
2905441
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Haft, Carol R
Project Start
1992-09-30
Project End
2000-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Lin-Cereghino, Geoffrey Paul; Godfrey, Laurie; de la Cruz, Bernard J et al. (2006) Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris. Mol Cell Biol 26:883-97
Cereghino, Geoff P Lin; Cereghino, Joan Lin; Ilgen, Christine et al. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329-32
Lin Cereghino, G P; Lin Cereghino, J; Sunga, A J et al. (2001) New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris. Gene 263:159-69
Lin Cereghino, G P; Sunga, A J; Lin Cereghino, J et al. (2001) Expression of foreign genes in the yeast Pichia pastoris. Genet Eng (N Y) 23:157-69
Johnson, M A; Snyder, W B; Cereghino, J L et al. (2001) Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins. Yeast 18:621-41
Cregg, J M; Cereghino, J L; Shi, J et al. (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23-52
Cereghino, J L; Cregg, J M (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45-66
Johnson, M A; Waterham, H R; Ksheminska, G P et al. (1999) Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris. Genetics 151:1379-91
Stasyk, O V; van der Klei, I J; Bellu, A R et al. (1999) A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy. Curr Genet 36:262-9
Cereghino, G P; Cregg, J M (1999) Applications of yeast in biotechnology: protein production and genetic analysis. Curr Opin Biotechnol 10:422-7

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