Pfister K K, Benashski S E, Dillman J F, Patel-King R S, King S M
Department of Cell Biology, University of Virginia Health Science Center, Charlottesville 22908-0439, USA.
Cell Motil Cytoskeleton. 1998;41(2):154-67. doi: 10.1002/(SICI)1097-0169(1998)41:2<154::AID-CM6>3.0.CO;2-E.
Intracellular transport along microtubules uses the motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein is responsible for movement to the minus ends of microtubules and the evidence indicates that dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to identify and clone the subunit polypeptides of these two complexes, in particular their light chains. Dynactin is made up of eight subunits of approximately 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide gel electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-Mr region of the gel was obtained and a candidate polypeptide identified by a screen of the databases. This polypeptide has a predicted molecular weight of 20,822 Da. Using an antibody to a different region of this protein, we demonstrate that it copurifies with microtubules and elutes from the microtubule pellet with characteristics similar to those of the dynactin complex and distinct from those of cytoplasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, but not with kinesin or cytoplasmic dynein. Together these results demonstrate that this polypeptide is the p24 subunit of dynactin. Analysis of the predicted amino acid sequence of p24 shows that it is a unique protein that has no significant similarity to known enzymes or other proteins. Structural analysis indicates that most of this protein will form an alpha-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynactin indicates that there is one molecule of p24 per dynactin complex, these characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex.
沿着微管的细胞内运输利用驱动蛋白胞质动力蛋白和驱动蛋白。胞质动力蛋白负责向微管负端的移动，证据表明动力蛋白与另一种蛋白复合物动力肌动蛋白相互作用。为了更好地理解这些蛋白质的功能，我们试图鉴定并克隆这两种复合物的亚基多肽，尤其是它们的轻链。动力肌动蛋白由八个亚基组成，分子量约为24,000至160,000道尔顿。为了克隆p24亚基，通过SDS聚丙烯酰胺凝胶电泳分离纯化的动力肌动蛋白的组分。获得了凝胶中24,000道尔顿区域胰蛋白酶肽的氨基酸序列，并通过数据库筛选鉴定了一种候选多肽。该多肽的预测分子量为20,822道尔顿。使用针对该蛋白不同区域的抗体，我们证明它与微管共纯化，并以与动力肌动蛋白复合物相似且与胞质动力蛋白不同的特性从微管沉淀中洗脱。该多肽在蔗糖密度梯度上与动力肌动蛋白共沉降，并且也与动力肌动蛋白共免疫沉淀，但不与驱动蛋白或胞质动力蛋白共免疫沉淀。这些结果共同表明该多肽是动力肌动蛋白的p24亚基。对p24预测氨基酸序列的分析表明，它是一种独特的蛋白质，与已知酶或其他蛋白质没有明显的相似性。结构分析表明，该蛋白的大部分将形成α-螺旋，并且分子的部分可能参与卷曲螺旋的形成。由于对动力肌动蛋白的化学计量分析表明每个动力肌动蛋白复合物中有一个p24分子，这些特性表明该多肽可能参与蛋白质-蛋白质相互作用，也许参与动力肌动蛋白复合物的组装。
