Using suppressor analysis to elucidate peroxisome biogenesis and peroxin function The import of matrix enzymes that catalyze metabolic reactions supporting growth and development is central to peroxisome biogenesis and function. The current model for peroxisomal matrix protein import provides a framework of peroxisomal proteins (peroxins) involved and the associations these peroxins make to import proteins into the peroxisome. However, this model lacks mechanistic details of the associations among these peroxins, contributions of non-transport peroxins to import, and possibly unidentified peroxins. This project proposes using mutant suppression screens to identify new peroxisomal components, reveal novel genetic interactions between known peroxins, and provide more detailed understanding of known associations. Mutations in distinct steps in matrix protein import were chosen for suppression screens: pex12-1, a mutant of a peroxin of the ubiquitin ligating complex that aids in peroxisomal receptor recycling, and pex14-1 and pex14-6, mutants of a docking complex peroxin that recruits peroxisomal receptors and aids in matrix protein insertion. In each screen, the initial mutant (pex12-1, pex14-1, or pex14-6) was mutagenized and then screened for lines with beneficial secondary mutations that alleviated one or more peroxisomal defects. Numerous mutant suppressors already have been isolated through these screens. For each prioritized suppressor, this project aims to identify the mutation causing suppression through whole- genome sequencing, bioinformatics, and recombination mapping. Suppression mechanisms will be elucidated by combining different mutations and comparing impacts on peroxisome function by monitoring physiological, cell biological, and molecular responses. Characterizing the novel mutations identified in these screens and determining the mechanisms through which they restore or bypass peroxisome dysfunction will elucidate the roles of the suppressor gene and the targeted peroxin in peroxisome function and refine our understanding of matrix protein import. Furthermore, because this project is designed to uncover means of repairing or circumventing peroxisomal dysfunction in mutants of PEX12 and PEX14 and because peroxins often have conserved function among diverse species, the results of this proposal may inform therapies for peroxisome biogenesis disorder patients with mutations that impair docking (PEX14) or ubiquitination (PEX12) of receptor peroxins. !
Peroxisomes are organelles necessary for the survival and development of almost all multicellular organisms; peroxisomal defects cause severe degenerative disorders in humans. To properly support cellular metabolism, peroxisomes must efficiently import necessary matrix enzymes through the coordinated association of several complexes. The goal of this project is to improve our understanding of peroxisomal matrix protein import by uncovering novel peroxisomal components and providing mechanistic details of the peroxins and complexes involved.