Engelbrecht S, Deckers-Hebestreit G, Altendorf K, Junge W
Department of Biophysics, University of Osnabrück, Federal Republic of Germany.
Eur J Biochem. 1989 May 1;181(2):485-91. doi: 10.1111/j.1432-1033.1989.tb14750.x.
F0F1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The delta subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0. We wanted to know whether or not delta of one species was effective between F0 and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(-delta), and delta were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-delta)+chloroplast delta, EF1, EF1(-delta)+E. coli delta were also effective but to lesser extent. CF1(-delta)+E. coli delta and EF1(-delta)+chloroplast delta restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1 (-delta)+chloroplast delta and CF1(-delta)+E. coli delta gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-delta)+E. coli delta by energization of the vesicles with NADH, while Ef1(-delta)+chloroplast delta was ineffective. All 'mixed' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed F0 (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast delta and E. coli delta. It favors a role of delta as a conformational transducer rather than as a proton conductor between F0 and F1.
F0F1 - ATP合酶利用质子跨膜电化学势提供的自由能催化由ADP和Pi合成ATP。F1的δ亚基在通道部分F0和催化部分F1之间的界面处起重要作用。在叶绿体中，它可以堵塞CF0的质子传导，而在大肠杆菌中，它是EF1与EF0结合所必需的。我们想知道一个物种的δ亚基在另一个物种的F0和F1之间是否有效，反之亦然。为此，将各自的偶联膜（类囊体、大肠杆菌外翻囊泡）（部分）去除F1，并将纯化的F1、F1（-δ）和δ以各种组合添加到去除F1的膜中。通过吩嗪硫酸甲酯介导的循环光合磷酸化速率在类囊体中测量重组效率，通过9 - 氨基 - 6 - 氯 - 2 - 甲氧基吖啶荧光猝灭程度在大肠杆菌外翻囊泡中测量重组效率。向部分去除CF1的类囊体囊泡中添加CF1可最大程度地恢复光合磷酸化。CF1（-δ）+叶绿体δ、EF1以及EF1（-δ）+大肠杆菌δ也有效，但程度较小。CF1（-δ）+大肠杆菌δ和EF1（-δ）+叶绿体δ可将光合磷酸化恢复到较小但仍显著的程度。对于通过反复用EDTA处理大肠杆菌膜制备的去除F1的外翻囊泡，与用NADH使囊泡通电时的EF1或EF1（-δ）+大肠杆菌δ相比，添加CF1、CF1（-δ）+叶绿体δ和CF1（-δ）+大肠杆菌δ产生的9 - 氨基 - 6 - 氯 - 2 - 甲氧基吖啶荧光猝灭程度约为一半，而Ef1（-δ）+叶绿体δ无效。所有“混合”组合可能仅通过堵塞通过暴露的F0的质子泄漏（结构重组）而具有重组活性，而非通过催化活性。然而，鉴于叶绿体δ和大肠杆菌δ之间较弱的序列相似性，这种交叉重组令人惊讶。这支持了δ作为构象转换器而非F0和F1之间质子导体的作用。
