Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.
Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.
复制标题
DOI：
10.1083/jcb.134.2.315
复制
发表时间：
1996-07
期刊：
影响因子：
--
通讯作者：
Schnell DJ
中科院分区：
文献类型：
--
作者：
Ma Y;Kouranov A;LaSala SE;Schnell DJ
在蛋白质输入的早期阶段，利用化学交联策略研究了前体蛋白与叶绿体包膜组分的相互作用。在缺乏能量的情况下，在前体结合试验中，外包膜进口机制的两个组成部分IAP86和IAP75与核酮糖- 1,5 -二磷酸羧化酶（pS）的小亚基前体的转运序列交联。当存在支持最大前体与包膜结合的ATP或GTP浓度时，与传递序列的交联主要发生在IAP75和一个先前未识别的内膜21-kD多肽上，表明传递序列已经插入外膜。在ATP或GTP存在的情况下，囊膜组分与第二前体优选氧还蛋白成熟部分的序列发生交联，这表明在这些条件下，远离转运序列的序列被带到外膜附近。IAP75和第三个进口成分IAP34与溶解包膜的IAP86抗体共免疫沉淀，表明这三种蛋白在外膜形成稳定的复合物。基于这些观察结果，我们提出IAP86和IAP75作为多亚基复合物的组成部分，介导转运序列的能量独立识别和随后三磷酸核苷诱导的转运序列穿过外膜的插入。
The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross- linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.