肌球蛋白是与肌动蛋白细胞骨架相关的重要运动蛋白。在结构上,肌球蛋白作为异聚复合物,其中较小的轻链,如钙调蛋白(CaM),与颈部区域的异亮氨酸-谷氨酰胺(IQ)域结合以促进机械酶活性。我们最近鉴定了拟南芥CaM样(CML)蛋白CML13和CML14作为含有多个IQ域的蛋白质的相互作用者,包括肌球蛋白VIII.这里,我们证明了CaM,CML13和CML14结合所有四种拟南芥肌球蛋白VIII同工型的颈部区域。在测试与肌球蛋白VIIIs结合的CML中,CaM,CML13和CML14在植物分裂荧光素酶蛋白相互作用测定中给出最强的信号。体外,重组CaM,CML13和CML14显示出特异性,高亲和力,与肌球蛋白VIIIs的IQ结构域不依赖钙的结合。CaM,当与含有IQ和肌球蛋白VIIIs尾部结构域的红色荧光蛋白-肌球蛋白融合蛋白共表达时,CML13和CML14共定位在质膜结合的斑点上。使用重组肌球蛋白VIIIs的体外肌动蛋白运动试验表明,CaM,CML13和CML14用作轻链。使用RNA沉默抑制CML13或CML14表达导致下胚轴表型缩短,类似于在四重肌球蛋白突变体中观察到的,肌球蛋白vii4KO.总的来说,我们的数据表明拟南芥CML13和CML14是新的肌球蛋白VIII轻链。
Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.