In vitro reconstitution of cortical actin assembly sites in budding yeast.
In vitro reconstitution of cortical actin assembly sites in budding yeast.
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萌芽酵母中皮质肌动蛋白组装位点的体外重构。
DOI：
10.1083/jcb.138.1.95
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发表时间：
1997-07-14
影响因子：
7.8
通讯作者：
Li, R
中科院分区：
文献类型：
--
作者：
Lechler, T;Li, R
我们已经开发了一种生化方法，用于确定皮质肌动蛋白组装位点的组件在极化酵母细胞，基于透化细胞测定，我们建立了肌动蛋白组装在体外。先前的分析表明，与细胞皮层相关的活动促进了肌动蛋白在芽中的聚合。在通过化学处理失活后，该活性可以从细胞质提取物重建回到透化细胞。提取物的分级显示，重建取决于两个顺序作用的蛋白质因子。Bee 1是一种与Wiskott-Aldrich综合征蛋白具有序列同源性的皮质肌动蛋白细胞骨架蛋白，是重建的第一步所需的。这一发现，以及与bee 1无效突变相关的肌动蛋白组织中的严重缺陷，表明Bee 1蛋白在控制细胞皮层的肌动蛋白聚合中起直接作用。在复溶的第二步中起作用的因子已通过常规色谱法鉴定。它由一种新的蛋白质Pca 1组成。序列分析表明，Pca 1具有与含有SH 3结构域的蛋白质和磷脂相互作用的潜力。
We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.