Lund A A, Blum P H, Bhattramakki D, Elthon T E
School of Biological Sciences and the Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588-0118, USA.
Plant Physiol. 1998 Mar;116(3):1097-110. doi: 10.1104/pp.116.3.1097.
We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.
我们利用二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹法，鉴定了大肠杆菌分子伴侣DnaK（热休克蛋白70，HSP70）和GroEL（伴侣蛋白60，cpn60）在玉米（玉米自交系B73）线粒体中的同源物。在热胁迫（42℃处理4小时）期间，HSP70和cpn60蛋白水平没有显著变化。相比之下，两种22-kD蛋白（HSP22）的水平显著升高。我们制备了针对玉米HSP70、cpn60和HSP22的单克隆抗体。对这些单克隆抗体与叶绿体、细胞质和线粒体组分以及不同植物物种的交叉反应性进行了表征。研究了线粒体HSP22在诱导温度、诱导所需时间以及胁迫解除后降解所需时间方面的表达情况。在+13℃热胁迫5小时后，黄化幼苗线粒体中出现了最大程度的HSP22表达。热胁迫解除后，HSP22蛋白以约4小时的半衰期消失，恢复21小时后无法检测到。在持续热胁迫条件下，HSP22水平保持较高。克隆了玉米线粒体HSP22的cDNA，并利用表达序列标签数据库中的序列将其延伸至全长。序列分析表明，HSP22是植物小热休克蛋白超家族的成员。
