The long-term goal of this proposal is to understand the genetic regulation and assembly mechanism of the peroxisome, an organelle which plays an essential role in the synthesis and turnover of lipids as well as in the removal of toxic compounds, such as hydrogen peroxide, in eukaryotic cells. In a variety of yeasts and in certain animal and plant tissues, a very rapid and dramatic proliferation of peroxisomes can be elicited by developmental changes or by alterations in cellular metabolic activity. The ability of cells to build new or expand existing peroxisomes in response to specific physiological requirements raises several questions: How does the cell regulate peroxisome expression? And how does the cell target the delivery of newly synthesized peroxisomal components from their site of manufacture to their correct site of assembly and function? To answer these questions, a combined biochemical and genetic approach will be used to identify, clone and characterize genes required for the regulation and for the targeting and assembly of peroxisomal matrix and membrane precursors in the yeast, Saccharomyces cerevisiae.
The aims of this proposal are: (i) to examine the genetic regulation of peroxisomes in yeast mutants which are defective in glucose repression; (ii) to isolate peroxisome assembly mutants by a direct, positive genetic selection that demands cytoplasmic localization of an enzyme (histidinol dehydrogenase) to which a peroxisomal targeting signal has been appended; (iii) to examine mutants obtained in the genetic selection by biochemical and morphological methods to confirm that the chromosomal mutations result in mislocalization of peroxisomal matrix and/or membrane protein precursors to the cytoplasm; (iv) to test peroxisome and cytosol fractions prepared from mutant and wildtype cells for their ability to sustain post-translational import and processing of a peroxisomal precursor in a cell-free assay; (v) to clone genes required for peroxisome assembly by screening a yeast genomic DNA library for complementation of the peroxisome assembly mutations; and (vi) to analyze the cloned genes by DNA sequencing and to prepare monospecific, polyclonal antibodies for subsequent structural and functional studies of the gene products. Understanding peroxisome assembly is important because a group of human genetic diseases have been described in which defects in brain development and liver function have been traced to mutations impairing peroxisome assembly and function. Use of an integrated genetic and biochemical dissection of peroxisome biogenesis in yeast should extend fundamental knowledge about the identity, structure, and function of key components in the assembly process. The development of nucleic acid probes and specific antibodies will facilitate identification of the mammalian counterparts and elucidation of the molecular details of human genetic defects which can impair peroxisome assembly and function.
| Yu, C; Kennedy, N J; Chang, C C et al. (1996) Molecular cloning and characterization of two isoforms of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase. J Biol Chem 271:24157-63 |
