Ni Zhang-Lin, Shi Xiao-Bing, Wei Jia-Mian
Shanghai Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
Biochemistry. 2004 Mar 2;43(8):2272-8. doi: 10.1021/bi035954r.
Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector. In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1). The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel. None of the mutants impaired ATP hydrolysis by CF(1).
利用诱变技术构建了菠菜（Spinacia oleracea）叶绿体ATP合酶δ亚基的7个截短突变体，这些突变体分别从N端缺失5、11、17或35个氨基酸残基，或从C端缺失3、9或15个氨基酸残基。通过酵母双杂交系统研究了这些突变体与叶绿体ATP酶所有其他亚基之间的相互作用。结果表明，N端缺失主要影响δ亚基与CF(1)其他部分之间的相互作用，但对与CF(0)区段的相互作用影响不显著。相反，δ亚基的C端截短主要影响其与CF(0)区段的相互作用，而对与CF(1)其他部分的相互作用造成的损害较小。如C端缺失（9个残基）突变体驱动的最低表达所示，δ亚基C端结构域的构象似乎对截短更敏感。进一步研究表明，δ亚基的C端截短极大地损害了其在NaBr囊泡中恢复循环光合磷酸化的能力，而N端截短对δ亚基堵塞CF(0)通道的能力影响很小。这些突变体均未损害CF(1)的ATP水解能力。
