Using suppressor analysis to elucidate peroxisome biogenesis and peroxin function

项目摘要/摘要 应用抑制子分析阐明过氧化物体的生物发生和过氧化物素功能 催化支持生长和发育的代谢反应的基质酶的进口是 过氧酶体的生物发生和功能的中心。过氧化体基质蛋白进口的现行模型 提供了所涉及的过氧化物体蛋白(过氧化物素)的框架以及这些过氧化物素所产生的关联 将蛋白质输入到过氧化酶体中。然而，该模型缺乏关于关联的机械性细节 在这些过氧物中，非运输过氧物对进口的贡献，以及可能不明的过氧物。 该项目建议使用突变抑制屏幕来识别新的过氧化物体成分，揭示 已知的过氧物之间的新的遗传相互作用，并提供对已知的 联想。选择基质蛋白导入过程中不同步骤的突变进行抑制筛查： Pex12-1，泛素连接复合体的过氧素突变体，有助于过氧体受体的循环， 以及pex14-1和pex14-6，这是一种对接复合过氧化物素的突变体，它招募过氧化物体受体和 基质蛋白插入中的艾滋病。在每个筛选中，初始突变体(pex12-1、pex14-1或pex14-6)是 诱变，然后筛选出具有有益的二次突变的品系，这些突变缓解了一个或多个 过氧化体缺陷症。许多突变抑制子已经通过这些筛子被分离出来。为 每个优先的抑制者，这个项目的目的是通过整体识别导致抑制的突变 基因组测序、生物信息学和重组图谱。压制机制将是 通过结合不同的突变并通过监测比较对过氧化物体功能的影响来阐明 生理、细胞生物学和分子反应。 鉴定在这些筛查中发现的新突变，并通过 他们恢复或绕过了过氧化物体功能障碍将阐明抑制基因和 靶向过氧化物素在过氧酶体中的作用，并加深我们对基质蛋白输入的理解。此外， 因为这个项目旨在揭示修复或绕过过氧化酶体功能障碍的方法 PEX12和PEX14的突变体以及由于过氧化物素通常在不同物种中具有保守功能， 这一建议的结果可能会为有突变的过氧化物体生物发生障碍患者的治疗提供参考。 这损害了受体过氧化物素的对接(PEX14)或泛素化(PEX12)。 好了！

Project summary/Abstract 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. !