Yao X, Cheng L, Forte J G
Department of Molecular and Cell Biology, University of California, Berkeley, 94720, USA.
J Biol Chem. 1996 Mar 22;271(12):7224-9. doi: 10.1074/jbc.271.12.7224.
The highly related actin isoforms are thought to have different functions. We recently demonstrated a polarized distribution of actin isoforms in gastric parietal cells and association of gastric ezrin with the cytoplasmic beta-actin isoform (Yao, X., Chaponnier, C., Gabbiani, G., and Forte, J. G. (1995) Mol. Biol. Cell. 6, 541-557). Here we used ultrastructural immunocytochemistry to verify that beta-actin is located within canalicular microvilli and the apical cortex of parietal cells, similar to the localization reported for ezrin. Furthermore, we tested whether ezrin binds preferentially to cytoplasmic beta-actin compared with the skeletal muscle alpha-actin isoform. Purified cytoplasmic beta-actin (from erythrocytes) and skeletal alpha-actin were assembled with gastric ezrin. Co-sedimentation experiments showed that gastric ezrin selectively co-pelleted with the beta-actin isoform and only very poorly with alpha-actin. Binding of erythrocytic beta-actin to ezrin is saturable with a molar ratio of approximately 1:10 (ezrin:actin) and a dissociation constant approximately 4.6 x 10(-8) M. In addition, ezrin promoted pyrene-labeled actin assembly, with predominant effects on filament elongation and a distinct preference for beta-actin compared with alpha-actin. Given these isoform-selective associations, we speculate that actin isoforms might segregate into different functional domains and exert specificity by interacting with isoform-orientated binding proteins.
人们认为高度相关的肌动蛋白异构体具有不同的功能。我们最近证实了胃壁细胞中肌动蛋白异构体的极化分布以及胃埃兹蛋白与细胞质β-肌动蛋白异构体的关联（姚，X.，沙波尼耶，C.，加比亚尼，G.，和福特，J.G.（1995年）《分子生物学细胞》6，541 - 557）。在这里，我们使用超微结构免疫细胞化学来验证β-肌动蛋白位于壁细胞的小管微绒毛和顶端皮质内，这与报道的埃兹蛋白的定位相似。此外，我们测试了与骨骼肌α-肌动蛋白异构体相比，埃兹蛋白是否优先结合细胞质β-肌动蛋白。将纯化的细胞质β-肌动蛋白（来自红细胞）和骨骼肌α-肌动蛋白与胃埃兹蛋白组装在一起。共沉降实验表明，胃埃兹蛋白选择性地与β-肌动蛋白异构体共沉淀，而与α-肌动蛋白的共沉淀效果很差。红细胞β-肌动蛋白与埃兹蛋白的结合是可饱和的，摩尔比约为1:10（埃兹蛋白：肌动蛋白），解离常数约为4.6×10⁻⁸M。此外，埃兹蛋白促进了芘标记的肌动蛋白组装，对细丝伸长有主要影响，并且与α-肌动蛋白相比，对β-肌动蛋白有明显的偏好。鉴于这些异构体选择性关联，我们推测肌动蛋白异构体可能会分隔到不同的功能域，并通过与异构体定向结合蛋白相互作用发挥特异性作用。
