Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan.
Plant Physiol. 2012 Apr;158(4):1656-65. doi: 10.1104/pp.112.193300. Epub 2012 Feb 21.
Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ΔN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ΔN proteins did not associate with membranes and atHsp93V-ΔΝ failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ΔN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ΔN transgenic plants. Hsp93-ΔN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.
叶绿体 93kDa 热休克蛋白（Hsp93/ClpC），一种 Hsp100 家族成员，被认为在叶绿体中有多种功能，包括作为基质中 ClpP 蛋白酶的调节伴侣，并作为包膜中蛋白质转位器的运动组件。事实上，尽管 Hsp93 是一种可溶性基质蛋白，但其中一部分与内包膜结合。这种 Hsp93 膜结合的机制和功能意义尚未确定。在这里，我们通过创建各种缺失构建体来绘制与 Hsp93 膜结合相关的重要区域，并发现只有删除氨基末端结构域的 Hsp93-ΔN 构建体与膜的结合减少。当转化为拟南芥（Arabidopsis thaliana）时，大多数 atHsp93V-ΔN 蛋白不与膜结合，并且 atHsp93V-ΔΝ 不能弥补 hsp93V 敲除突变体的淡绿色和蛋白导入缺陷表型。残留在膜上的 atHsp93V-ΔN 与中央蛋白转位器成分 Tic110 的结合进一步减少。然而，叶绿体谷氨酰胺合成酶的降解，ClpP 蛋白酶的潜在底物，在 hsp93V 突变体或 atHSP93V-ΔN 转基因植物中不受影响。Hsp93-ΔN 也具有与全长 Hsp93 相同的 ATPase 活性。这些数据表明，Hsp93 通过其氨基末端结构域与内包膜的结合对于 Hsp93 在体内的功能很重要。
