Palani Saravanan, Koester Darius, Balasubramanian Mohan K
Centre for Mechanochemical Cell Biology, Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK.
Wellcome Open Res. 2020 Jul 7;5:161. doi: 10.12688/wellcomeopenres.16082.1. eCollection 2020.
Tropomyosins are coiled-coil proteins that regulate the stability and / or function of actin cytoskeleton in muscle and non-muscle cells through direct binding of actin filaments. Recently, using the fission yeast, we discovered a new mechanism by which phosphorylation of serine 125 of tropomyosin (Cdc8), reduced its affinity for actin filaments thereby providing access for the actin severing protein Adf1/Cofilin to actin filaments causing instability of actin filaments. Here we use a genetic code expansion strategy to directly examine this conclusion. We produced in Cdc8-tropomyosin bearing a phosphate group on Serine-125 (Cdc8 ), using an orthogonal tRNA-tRNA synthetase pair that directly incorporates phosphoserine into proteins in response to a UAG codon in the corresponding mRNA. We show using total internal reflection (TIRF) microscopy that, whereas produced Cdc8 does not bind actin filaments, Cdc8 incubated with lambda phosphatase binds actin filaments. This work directly demonstrates that a phosphate moiety present on serine 125 leads to decreased affinity of Cdc8-tropomyosin for actin filaments. We also extend the work to demonstrate the usefulness of the genetic code expansion approach in imaging actin cytoskeletal components.
原肌球蛋白是一种卷曲螺旋蛋白，通过与肌动蛋白丝直接结合来调节肌肉细胞和非肌肉细胞中肌动蛋白细胞骨架的稳定性和/或功能。最近，我们利用裂殖酵母发现了一种新机制，即原肌球蛋白（Cdc8）丝氨酸125位点的磷酸化降低了其与肌动蛋白丝的亲和力，从而使肌动蛋白切断蛋白Adf1/丝切蛋白能够接触到肌动蛋白丝，导致肌动蛋白丝不稳定。在这里，我们使用遗传密码扩展策略直接验证这一结论。我们利用一对正交的tRNA-氨酰tRNA合成酶，通过在相应mRNA中的UAG密码子处直接将磷酸丝氨酸掺入蛋白质中，在丝氨酸125位点带有磷酸基团的Cdc8-原肌球蛋白（Cdc8 ）中进行表达。我们使用全内反射（TIRF）显微镜显示，虽然表达的Cdc8 不与肌动蛋白丝结合，但与λ磷酸酶孵育后的Cdc8 能与肌动蛋白丝结合。这项工作直接证明了丝氨酸125位点上的磷酸基团会导致Cdc8-原肌球蛋白与肌动蛋白丝的亲和力降低。我们还拓展了这项工作，以证明遗传密码扩展方法在成像肌动蛋白细胞骨架成分方面的实用性。
