Function and sorting of yeast peroxisomal membrane proteins

本课题以甲基营养型酵母Candida boidinii为模式生物，分析了过氧化物酶体膜蛋白（PMP）的生化功能与过氧化物酶体组装的关系，以及PMP的分选信号，通过分析PMP的过氧化物酶体基质和膜蛋白的表达调控，我们的结论是PMP的诱导发生在基质酶之前，并且这主要在mRNA水平上调节。克隆了博伊丁氏梭菌的PMP 20、PMP 30、PMP 47基因，并对其进行了基因敲除，分析了敲除菌株（pmp 30 DELTA和pmp 47 DELTA）对几种碳源的生长活性。这两种菌株都能在油酸和D-丙氨酸上生长，但不能在甲醇上生长，这些生长底物需要过氧化物酶体的代谢.电镜观察结果表明，Pmp 30 DELTA能直接促进过氧化物酶体的增殖，并影响细胞器的大小和形状.我们还证实了Pmp 30是同源的 ...更多信息 通过遗传互补实验，将pmp 47 Δ转化为酿酒酵母Pmp 27，并对pmp 47 Δ甲醇诱导细胞进行了详细的研究。二羟丙酮合酶（DHAS），这是一种甲醇诱导的过氧化物酶体基质酶的活性，在pmp 47 DELTA中未检测到。进一步的生物化学和免疫细胞化学实验显示DHAS蛋白以包涵体的形式聚集在细胞质中。然后，破坏两个过氧化物酶体缺陷突变体的PMP 47基因。在这些突变菌株中，DHAS具有酶活性，并存在于细胞质中。因此，我们提出了一个机制，即一个未知的小分子，通过Pmp 47的运输，是必需的DHAS在过氧化物酶体中的折叠或易位机制。接下来，我们分析了过氧化物酶体膜整合蛋白Pmp 47的靶向序列，它跨越了6次膜。我们证明了20个氨基酸的环，预测面对的矩阵，是必要的和足够的过氧化物酶体的排序。这是第一次报告的排序序列，称为mPTS，过氧化物酶体的膜蛋白。少

In this project, we analyzed the relation between the biochemical functions of peroxisomal membrane proteins (PMP) and the assembly of peroxisomes, and the sorting signal for PMP,dealing with the methlotrophic yeast Candida boidinii as a model organism.By the analysis of regulation of the expression of peroxisomal matrix and membrane proteins, we conclude that the induction of PMP occurs before the matrix enzymes and that this is regulated mainly at the mRNA level. We cloned the PMP20, PMP30, PMP47 gene from C.boidinii, disrupted these genes, and analyzed growing activity of the disruption strains (pmp30DELTA and pmp47DELTA) on several carbon sources. Both strains could grow on oleate and D-alanine, but not on methanol ; these growth substrates require peroxisomal metabolism.The results from the electron microscopic observation of pmp30DELTA showed that Pmp30 directly promotes peroxisome proliferation and affects organelle size and shape. And also we confirmed that Pmp30 is homologous … More to Pmp27 from Saccharomyces cerevisiae by genetic complementation experiments.Methanol-induced cells of pmp47DELTA were investicated in detail. The activity of dihydroxyacetone synthase (DHAS), which is a methanol-induced peroxisome matrix enzyme, was not detected in pmp47DELTA. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body. Then, the PMP47 gene of two peroxisome-deficient mutants were disrupted. In these mutant strains, DHAS was enzymatically active and found in the cytoplasm. So we propose a mechanism that an unknown small molcule, which transports by Pmp47, is necessary for the folding or the translocation machinery of DHAS within peroxisomes.Next, we analyzed the targeting sequence for peroxisomal integral membrane protein Pmp47, which spans the membrane six times. We demonstrated that the 20 amino acid loop, which predicted to face the matrix, is necessary and sufficient for sorting to peroxisomes. This is the first report of a sorting sequence, termed mPTS,for a peroxisomal integral membrane protein. Less