Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure.
Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure.
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DOI：
10.1083/jcb.119.1.153
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发表时间：
1992-10
期刊：
影响因子：
--
通讯作者：
Tabak HF
中科院分区：
文献类型：
--
作者：
Van der Leij I;Van den Berg M;Boot R;Franse M;Distel B;Tabak HF
我们已经开发了一个积极的选择系统，用于分离酿酒酵母过氧化物酶体功能紊乱的突变体。该选择基于在脂肪酸的过氧化物酶体β-氧化期间在野生型细胞中产生的过氧化氢（H2 O2）的致死性。总之，17个突变体过氧化物酶体生物合成的一般损害被隔离，揭示了他们无法生长的油酸作为唯一的碳源和他们的异常细胞分馏模式的过氧化物酶体酶。突变体被证明具有单基因缺陷，并落入12个互补组。使用EM通过免疫细胞化学对每个互补组的代表成员进行形态学检查。在一个突变体中，过氧化物酶体的诱导和形态是正常的，但硫解酶的进口被废除，而在另一个形态不同于野生型：堆叠的过氧化物酶体膜存在，能够进口硫解酶，但不能过氧化氢酶。这些突变体表明过氧化物酶体蛋白进口涉及的多个组件的存在。一些突变体表现出葡萄糖抑制细胞的表型特征，表明信号转导途径中断，导致细胞器增殖。在剩余的突变体中，形态学上可检测的过氧化物酶体是不存在的：这种表型也是从患有齐薇格综合征（Zellweger syndrome）的患者的成纤维细胞中已知的，齐薇格综合征是一种由过氧化物酶体损伤引起的病症。
We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta- oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.