BIOGENESIS OF PEROXISOME WITH YEAST MUTANTS AND CLONED GENES

Zellweger综合征引起的主要缺陷与过氧化体组装受损有关。到目前为止，该综合征的病因可分为9个遗传互补组。对于它们中的任何一个，基因产物的功能都还没有被理解。在这项联合研究中，分离了过氧化体组装所必需的酵母基因，以研究该细胞器的生物发生过程。分离到了过氧化体组装缺陷的酿酒酵母突变株，该突变株能够在醋酸盐上生长，但在油酸上生长，胞质中的前体基质酶错位。突变体有18个互补基团(pas1-pas18)。PAS1基因编码一个117 kDa的疏水性蛋白质。预测的序列包含两个假定的ATP结合位点，并揭示了与参与多种细胞过程的蛋白质的结构关系：细胞周期控制(CDC48P)、囊泡介导的蛋白运输(Sec18P)和HIV的基因表达(TBP-1)。PAS2基因的产物是泛素结合蛋白家族的第10个成员。Pas2突变细胞积累了含有pas3p的膜结构(见下文)。这些结构可能是所谓的齐薇格成纤维细胞的幽灵。PAS3基因编码的一种50.6 kDa的PAS3P蛋白被认为是通过其氨基末端被锚定在过氧化体膜上的，而该分子的大部分暴露在胞浆中。缺乏PAS3野生型等位基因的细胞在过氧化物酶的分布方面表现出与pas1突变体相同的表型，并对有效的活性氧增殖剂白花蛇舌素敏感。

The primary defect arising from Zellweger syndrome is linked to impaired assembly of peroxisomes. The causes of the syndrome are thus far classified into 9 genetic complementation groups. For none of them, the function of the gene product has been understood. In this joint Research, yeast genes essential for the assembly of peroxisomes were isolated to study the process of the biogenesis of this organelle.Sacchromyces cerevisiae mutants defective in peroxisome assembly were isolated that were able to grow on acetate but on oleic acid, and mislocated proxisomal matrix enzymes in the cytosol. There are 18 complementation groups of mutants (pas1-pas18). The PAS1 gene encoded a hydrophobic 117-kDa protein. The predicted sequence contained two putative ATP-binding sites and revealed a structural relationship to proteins involved in various cellular processes : control of cell cycle (Cdc48p), vesicle-mediated protein transport(Sec18p), and gene expression of HIV (TBP-1). The product of the PAS2 gene was the 10th member of the ubiquitin-conjugating protein family. The pas2 mutant cells accumulated the membranous structures that contained pas3p (see below). These structures could be the so-called ghosts of Zellweger fibroblasts. Pas3p, a 50.6-kDa protein encoded by the PAS3 gene, was supposed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule was exposed to cytosol.The PAS1 homologue of a fatty-acid-assimilating yeast, Candida maltosa, was isolated. The cells lacking the wild-type alleles of PAS3 showed the same phenotype as the pas1 mutant in respect of the distribution of peroxisomal enzymes, and became sensitive to plumbagin, which is a potent propagator of reactive oxygen.