Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, 13210, USA.
Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, 13210, USA.
Exp Cell Res. 2019 Nov 15;384(2):111625. doi: 10.1016/j.yexcr.2019.111625. Epub 2019 Sep 19.
In both unicellular and multicellular organisms, long-tailed class I myosins function in clathrin-mediated endocytosis. Myosin 1e (Myo1e) in vertebrates and Myo1 in fission yeast have similar domain organization, yet whether these proteins or their individual protein domains are functionally interchangeable remains unknown. In an effort to assess functional conservation of class I myosins, we tested whether human Myo1e could replace Myo1 in fission yeast Schizosaccharomyces pombe and found that it was unable to substitute for yeast Myo1. To determine if any individual protein domain is responsible for the inability of Myo1e to function in yeast, we created human-yeast myosin-I chimeras. By functionally testing these chimeric myosins in vivo, we concluded that the Myo1e motor domain is unable to function in yeast, even when combined with the yeast Myo1 tail and a full complement of yeast regulatory light chains. Conversely, the Myo1e tail, when attached to the yeast Myo1 motor domain, supports localization to endocytic actin patches and partially rescues the endocytosis defect in myo1Δ cells. Further dissection showed that both the TH1 and TH2-SH3 domains in the human Myo1e tail are required for localization and function of chimeric myosin-I at endocytic sites. Overall, this study provides insights into the role of individual myosin-I domains, expands the utility of fission yeast as a simple model system to study the effects of disease-associated MYO1E mutations, and supports a model of co-evolution between a myosin motor and its actin track.
在单细胞和多细胞生物中，长尾 I 型肌球蛋白在网格蛋白介导的内吞作用中发挥作用。脊椎动物的肌球蛋白 1e（Myo1e）和裂殖酵母的 Myo1 具有相似的结构域组织，但这些蛋白质或其单个蛋白结构域是否在功能上可互换尚不清楚。为了评估 I 型肌球蛋白的功能保守性，我们测试了人源 Myo1e 是否可以替代裂殖酵母 Schizosaccharomyces pombe 中的 Myo1，但发现它无法替代酵母 Myo1。为了确定是否存在任何单个蛋白结构域导致 Myo1e 无法在酵母中发挥作用，我们创建了人-酵母肌球蛋白 I 嵌合体。通过在体内对这些嵌合肌球蛋白进行功能测试，我们得出结论，Myo1e 肌球蛋白结构域即使与酵母 Myo1 尾部和完整的酵母调节轻链结合，也无法在酵母中发挥作用。相反，当 Myo1e 尾部连接到酵母 Myo1 肌球蛋白结构域时，它支持定位到内吞作用的肌动蛋白斑，并部分挽救了 myo1Δ 细胞中的内吞缺陷。进一步的剖析表明，人源 Myo1e 尾部的 TH1 和 TH2-SH3 结构域对于嵌合肌球蛋白-I 在内吞作用部位的定位和功能都是必需的。总体而言，这项研究深入了解了单个肌球蛋白 I 结构域的作用，扩展了裂殖酵母作为研究与疾病相关的 MYO1E 突变影响的简单模型系统的用途，并支持肌球蛋白结构域与其肌动蛋白轨道之间共同进化的模型。
