Zhang L, Paakkarinen V, van Wijk K J, Aro E M
Department of Biology, University of Turku, FIN-20520 Turku, Finland.
J Biol Chem. 1999 Jun 4;274(23):16062-7. doi: 10.1074/jbc.274.23.16062.
Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.
多亚基膜蛋白复合物的组装过程目前还知之甚少。在本研究中，我们提供了直接证据，表明多跨膜蛋白D1蛋白在共翻译过程中组装成大型膜结合复合物——光系统II。在完整叶绿体的脉冲追踪实验中，D1蛋白的掺入过程中，类囊体膜中并未出现游离标记蛋白的短暂积累，并且光系统II亚复合物包含17 kDa、22 kDa和25 kDa的新生D1中间体。这些N端D1中间体可以与抗D2蛋白的抗血清进行共免疫沉淀，这表明D1蛋白在共翻译过程中组装到PS II复合物中。通过分析短脉冲标记后从类囊体膜中释放的核糖体新生链复合物，获得了D1蛋白在共翻译过程中组装到光系统II的进一步证据。在非变性条件下，用针对D1、D2蛋白以及CP47的抗血清可以免疫沉淀放射性标记的D1中间体。然而，当核糖体沉淀用SDS溶解时，这些中间体与CP47的相互作用完全丧失，但25 kDa的D1中间体与D2蛋白之间仍保持强烈相互作用。综上所述，我们的结果表明，在光系统II的修复过程中，新合成的D1蛋白在共翻译过程中组装到光系统II中，这涉及新生D1链与D2蛋白的直接相互作用。
