Wu C, Seibert F S, Ko K
Department of Biology, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1994 Dec 23;269(51):32264-71.
Translocation intermediates of the chimeric protein precursor Oee1-Dhfr were generated and used to identify envelope components in close proximity to the arrested precursor. The translocation of Oee1-Dhfr across the chloroplast envelope can be arrested at low ATP levels or by prebinding the fusion precursor with anti-Dhfr IgGs. The arrested Oee1-Dhfr precursor appears to span both the outer and inner envelope membranes. Translocational arrest of Oee1-Dhfr by low ATP levels was reversible, and import was restored upon resupplementation with higher ATP levels. Chemical cross-linking and co-immunoprecipitation with monospecific antibodies indicate that two outer envelope membrane proteins (Com44 and Com70) and at least one inner envelope protein (Cim44 and Cim97) were found to be in close proximity to Oee1-Dhfr during translocation. The Com70 protein was further studied and additional evidence for its role in chloroplast protein import is presented.
嵌合蛋白前体Oee1-Dhfr的易位中间体被生成，并用于识别与停滞的前体紧密相邻的包膜成分。Oee1-Dhfr穿过叶绿体包膜的易位可在低ATP水平下被阻止，或通过用抗Dhfr IgG预先结合融合前体来实现。停滞的Oee1-Dhfr前体似乎跨越了外膜和内膜。低ATP水平对Oee1-Dhfr的易位停滞是可逆的，在补充更高水平的ATP后，导入得以恢复。化学交联和用单特异性抗体进行的共免疫沉淀表明，在易位过程中发现两种外膜蛋白（Com44和Com70）和至少一种内膜蛋白（Cim44和Cim97）与Oee1-Dhfr紧密相邻。对Com70蛋白进行了进一步研究，并提供了其在叶绿体蛋白导入中作用的额外证据。
