Liu B, Cyr R J, Palevitz B A
Department of Botany, University of Georgia, Athens 30602, USA.
Plant Cell. 1996 Jan;8(1):119-132. doi: 10.1105/tpc.8.1.119.
The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development. However, relatively little is known about plant KLPs. We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide. Such KLPs typically move materials toward the minus end of microtubules. An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts. During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division. Staining was concentrated near the midzone during metaphase and was retained there during anaphase. The phragmoplast was also stained. Similar localization patterns were seen in tobacco BY-2 cells. The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP). A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass. When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor. Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody. Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast. A similar protein is also present in other species.
驱动蛋白样蛋白（KLPs）是一个大家族的正向或负向微管马达蛋白，在细胞内运输、有丝分裂、减数分裂和发育过程中起着重要作用。然而，人们对植物KLPs的了解相对较少。我们制备了一种针对拟南芥KLP（KatAp）微管结合域中两个肽段的抗体，KatAp由KatA基因编码，KatA基因家族的驱动域位于多肽的C末端附近。这类KLPs通常将物质向微管的负端运输。在拟南芥幼苗提取物的免疫印迹上，用该抗体检测到了一条与KatAp相对应的免疫反应条带（分子量为140,000）。在免疫荧光定位过程中，该抗体在拟南芥间期悬浮细胞的细胞质和细胞核中产生微弱、可变的染色，但在分裂期对有丝分裂装置的染色要强得多。在中期，染色集中在中区附近，并在后期保留在那里。成膜体也被染色。在烟草BY-2细胞中也观察到了类似的定位模式。根据富集KLPs的程序（在AMP-PNP存在下而非ATP存在下与微管结合）制备的小鼠脑部分中，该抗体产生了一条单带（分子量为130,000）。来自胡萝卜悬浮细胞的类似部分产生了一条具有相似表观分子量的交叉反应多肽。当在紫杉醇和ATP存在下裂解分裂的BY-2细胞时，抗体染色迅速向两极移动，这支持了负端马达蛋白的存在。没有ATP、有AMP-PNP或有ATP加抗体时均未发生移动。我们的结果表明，KatA编码的蛋白KatAp在拟南芥中表达，并特异性定位于有丝分裂装置和成膜体的中区。其他物种中也存在类似的蛋白。
