Division of Applied Life Science (BK21 and WCU Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.
Plant Physiol Biochem. 2012 Sep;58:29-36. doi: 10.1016/j.plaphy.2012.06.006. Epub 2012 Jun 17.
When plants are exposed to extreme temperature, stress-inducible proteins are highly induced and involved in subcellular defence mechanisms. Hsp70, one of stress-inducible proteins, functions as an ATP-dependent molecular chaperone in broad organisms to process such as the inhibition of protein denaturation, promotion of protein folding, and renaturation of denatured proteins. In this study, we isolated a heat-inducible orchardgrass Hsp70 (DgHsp70) that is a homolog of cytosolic Hsp70 that possesses a CaM-binding domain. Purified DgHsp70 protein displayed dose-dependent ATPase, holdase, and ATP-dependent foldase activities. To investigate functional roles of DgHsp70 by the association of Arabidopsis calmodulin-2 (AtCaM2), showing heat-sensitive reduction on transcription, we first characterized the binding activity by gel-overlay assay. DgHsp70 binds to AtCaM2 in the presence of Ca(2+) via a conserved CaM-binding domain. Ca(2+)/AtCaM2 binding decreased ATPase activity of DgHsp70, and concomitantly, reduced foldase activity. Based on the protein structure of bovine Hsc70, which is the closest structural homolog of DgHsp70, a CaM-binding domain is located near the ATP-binding site and CaM may span the ATP-binding pocket of Hsp70. Its decreased functional foldase activity may be caused by blocking ATP hydrolysis after Ca(2+)/AtCaM2 binding. It may associate with inhibition of functional activity of DgHsp70 in the absence of stress and/or de novo protein synthesis of DgHsp70 in the presence of thermal stress condition.
当植物暴露在极端温度下时，应激诱导蛋白会被高度诱导，并参与细胞防御机制。应激诱导蛋白之一的 Hsp70 在广泛的生物体中作为一种依赖 ATP 的分子伴侣发挥作用，以处理蛋白质变性的抑制、促进蛋白质折叠和变性蛋白质的复性等过程。在这项研究中，我们分离了一种热诱导的果园草 Hsp70（DgHsp70），它是细胞质 Hsp70 的同源物，具有 CaM 结合域。纯化的 DgHsp70 蛋白表现出剂量依赖性的 ATPase、holdase 和 ATP 依赖性的折叠酶活性。为了通过与拟南芥钙调蛋白-2（AtCaM2）的结合来研究 DgHsp70 的功能作用，显示出转录的热敏感降低，我们首先通过凝胶覆盖测定来表征结合活性。在 Ca2+存在下，DgHsp70 通过保守的 CaM 结合域与 AtCaM2 结合。Ca2+/AtCaM2 结合降低了 DgHsp70 的 ATPase 活性，同时降低了折叠酶活性。基于与 DgHsp70 结构最接近的牛 Hsc70 的蛋白质结构，CaM 结合域位于 ATP 结合位点附近，CaM 可能横跨 Hsp70 的 ATP 结合口袋。其降低的功能折叠酶活性可能是由于 Ca2+/AtCaM2 结合后阻止了 ATP 水解。它可能与应激条件下缺乏功能活性的 DgHsp70 以及热应激条件下缺乏新合成的 DgHsp70 的功能活性抑制有关。
