Wetzel C. M., McCarty R. E.
Field of Botany, Cornell University, Ithaca, New York 14853 (C.M.W.).
Plant Physiol. 1993 May;102(1):241-249. doi: 10.1104/pp.102.1.241.
A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids.
为了研究CF1与亚基III之间的相互作用，对叶绿体ATP合酶复合体（CF1[叶绿体偶联因子1] - CF0[叶绿体ATP合酶的跨膜部分]）进行了纯化，该复合体除了亚基III（也称为蛋白脂质亚基）外，所有CF0亚基均已缺失。亚基III在光合磷酸化过程中质子跨类囊体膜转运中具有假定作用；因此，涉及亚基III的亚基相互作用的精确模型对于阐明能量偶联的机制和调节将是有价值的。通过阴离子交换色谱期间的去污剂处理，从菠菜（Spinacia oleracea）类囊体的粗CF1 - CF0制剂中纯化该复合体。通过氨基酸分析和N端测序对复合体中的亚基III进行了阳性鉴定。通过线性蔗糖梯度离心、免疫沉淀以及将复合体掺入大豆卵磷脂脂质体中，验证了亚基III与CF1的结合。掺入脂质体后，通过乙二胺四乙酸处理从CF1 - III复合体中去除CF1。亚基III - 蛋白脂质体能够重新结合纯化的CF1。这些结果表明，在菠菜类囊体中，亚基III直接与CF1相互作用。
