Bennoun P, Atteia A, Pierre Y, Delosme M
Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Associée 1187, Paris, France.
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10202-6. doi: 10.1073/pnas.92.22.10202.
We have investigated a light-conditional mutant of Chlamydomonas reinhardtii (J12) that is unable to synthesize chlorophyll in the dark with the aim of characterizing the mitochondrial membrane polypeptides of this alga. A crude membrane fraction derived from etiolated cells was analyzed by gel electrophoresis, immunoblot analysis, and pulse-labeling in the presence of specific protein synthesis inhibitors. This fraction contained both mitochondrial and etioplast membranes, and the latter contained appreciable amounts of subunits of the cytochrome b6f complex. The mitochondria-encoded subunit 1 of cytochrome-c oxidase called COX1 was identified, and its synthesis was detected in this membrane fraction. The redox-difference spectra of mitochondrial cytochromes were studied in whole cells and membrane fractions, in both respiratory-competent and -deficient strains. Mitochondrial membranes could be further purified after sucrose gradient centrifugation. The use of etiolated cells and their membrane extracts, in association with appropriate methodologies, opens ways to study the molecular genetics of mitochondria in C. reinhardtii and allows us to address the question of the cooperation established between the three genetic compartments of a plant cell.
我们研究了莱茵衣藻的一种光条件突变体（J12），该突变体在黑暗中无法合成叶绿素，目的是表征这种藻类的线粒体膜多肽。通过凝胶电泳、免疫印迹分析以及在特定蛋白质合成抑制剂存在下的脉冲标记，对来自黄化细胞的粗膜组分进行了分析。该组分包含线粒体膜和黄化质体膜，后者含有可观数量的细胞色素b6f复合物亚基。鉴定出了线粒体编码的细胞色素c氧化酶亚基1（称为COX1），并在该膜组分中检测到了其合成。在有呼吸能力和呼吸缺陷的菌株中，对全细胞和膜组分中的线粒体细胞色素的氧化还原差异光谱进行了研究。线粒体膜可在蔗糖梯度离心后进一步纯化。使用黄化细胞及其膜提取物，并结合适当的方法，为研究莱茵衣藻线粒体的分子遗传学开辟了道路，并使我们能够解决植物细胞三个遗传区室之间建立的合作问题。
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