Peroxisomes are essential organelles that are found in virtually all eukaryotic cells. They catalyze a variety of oxidations, metabolic interconversions and biosynthetic reactions. Defects in peroxisomal assembly in humans are the coarse of the catastrophic childhood diseases Zellweger syndrome and neonatal adrenoleukodystrophy which lead to death at an early age. Although much is now known about the functions of peroxisomes, considerably less is understood about the assembly of these organelles. The long-term goal of this research is the understanding of the molecular interactions responsible for the targeting, membrane translocation or assembly, and maturation of peroxisomal proteins. Two yeast species Candida boidinii and Saccharomyces cerevisiae will be utilized for their strengths in characterizing three aspects of peroxisomal assembly. (1) Targeting sequences on a peroxisomal integral membrane protein, PMP47, will be identified. Fusions of this protein from the methylotrophic yeast C boidinii, and dihydrofolate reductase will be constructed. Their sorting in vivo into the peroxisomal membrane of S. cerevisiae will be monitored by techniques of cell fractionation and immunoblotting, and immunomicroscopy. Nested deletions in the PMP47 part of the fusions will ge generated to identify putative targeting sequences. Such sequences will be confirmed in separate constructions. Allowed alterations of the targeting signal(s) will be determined. Antibodies against PMP47 and dihydrofolate reductase will be used in these studies. (2) The function of BP48, a protein that specifically and competitively binds to a peroxisomal targeting sequence in vitro, will be analyzed. BP48 will be purified by affinity chromatography and antibodies will be raised against it. These will be used to localize the protein within the cell. A pulse-chase and co-immunoprecipitation protocol, using the BP48 antibodies will be performed to determine if binding to newly synthesized peroxisomal proteins occurs in vivo. The corresponding gene will be isolated and a disruption strain in S. cerevisiae will be constructed to further assess the importance of this protein in peroxisomal assembly. (3) The function of the three abundant peroxisomal membrane proteins in S. cerevisiae, scPMP24, scPMP31, and scPMP32, will be proved. No functions have yet been shown for any specific bona fide peroxisomal integral membrane protein. The genes encoding these proteins will be cloned wither using peptide sequences from the purified proteins or by homology to proteins of similar size in peroxisomal membranes of C. Boidinii. Clues to function of the scPMPs may be obtained from the inferred primary structures. Strains containing the disrupted genes, if viable, will be studied by a variety of enzymatic, morphological, and biosynthetic assays to assess the role of these proteins on peroxisomal assembly and function. If appropriate, the genes will be placed on a regulated promoter to study effects of different levels of expression on function. If no phenotype is observed, strains containing a combination of disruptions will be generated to test for redundant functions. Ultimately, hypotheses of function will be tested in reconstituted systems. The genes will also be useful for future studies of the assembly and topology of this class of proteins.
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