Myosin surface loop 4 modulates inhibition of actomyosin 1b ATPase activity by tropomyosin.
Myosin surface loop 4 modulates inhibition of actomyosin 1b ATPase activity by tropomyosin.
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肌球蛋白表面环 4 调节原肌球蛋白对肌动球蛋白 1b ATP 酶活性的抑制。
DOI：
10.1021/bi602439f
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Coluccio,LynneM
中科院分区：
文献类型：
--
作者：
Lieto-Trivedi,Alena;Dash,Sheffali;Coluccio,LynneM
对I类肌球蛋白（MyoE）的结构研究预测，环4（靠近肌动蛋白结合区的表面环，I类肌球蛋白比其他肌球蛋白亚类长）可能会限制肌动蛋白结合蛋白（如原肌球蛋白）时肌球蛋白I与肌动蛋白的结合，并可能解释肌球蛋白I被排除在应力纤维之外的原因。为了验证这些假设，突变体分子的相关哺乳动物I类肌球蛋白，Myo 1b，其中环4被截断（从氨基酸序列RMNGLDES NGLD）或替换为较短的和不同的环4 inDictyosteliummyosin II（GAGEGA），在体外表达和他们的相互作用与肌动蛋白和肌动蛋白原肌球蛋白进行了测试。饱和量的表达成纤维细胞原肌球蛋白-2导致野生型Myo 1b的最大肌动蛋白激活的Mg 2 +-ATP酶活性降低，但对两种突变体的肌动蛋白激活的Mg 2 +-ATP酶活性几乎没有影响。在运动性测定中，当存在原肌球蛋白-2时，很少有肌动蛋白丝与Myo 1b-WT包被的盖玻片紧密结合，而在存在和不存在原肌球蛋白-2的情况下，肌动蛋白丝都与Myo 1b-NGLD或Myo 1b-GAGEGA结合并移位。当在哺乳动物细胞中表达时，像野生型一样，突变肌球蛋白在很大程度上被排除在含有原肌球蛋白的肌动蛋白丝之外，这表明在细胞中除了环4之外的其他因素决定肌球蛋白I靶向肌动蛋白丝的特定亚群。
Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from stress fibers. To test these hypotheses, mutant molecules of the related mammalian class I myosin, Myo1b, in which loop 4 was truncated (from an amino acid sequence of RMNGLDES to NGLD) or replaced with the shorter and distinct loop 4 found inDictyosteliummyosin II (GAGEGA), were expressed in vitro and their interaction with actin and with actin-tropomyosin was tested. Saturating amounts of expressed fibroblast tropomyosin-2 resulted in a decrease in the maximum actin-activated Mg2+-ATPase activity of wild-type Myo1b but had little or no effect on the actin-activated Mg2+-ATPase activity of the two mutants. In motility assays, few actin filaments bound tightly to Myo1b-WT-coated cover slips when tropomyosin-2 was present, whereas actin filaments both bound and were translocated by Myo1b-NGLD or Myo1b-GAGEGA in both the presence and absence of tropomyosin-2. When expressed in mammalian cells, like the wild type, the mutant myosins were largely excluded from tropomyosin-containing actin filaments, indicating that in the cell additional factors besides loop 4 determine targeting of myosins I to specific subpopulations of actin filaments.