Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA.
EMBO J. 2010 Mar 17;29(6):1033-44. doi: 10.1038/emboj.2010.5. Epub 2010 Feb 11.
During clathrin-mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin-binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile alpha-motif (SAM) domain and shows a propensity to oligomerize. By co-immunoprecipitation, Sla1p binds to clathrin and self-associates in vivo. Mutations in the clathrin-binding motif that abolish clathrin binding and structure-based mutations in SHD2 that impede self-association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin-mediated endocytosis.
在网格蛋白介导的胞吞作用中，衔接蛋白在协调网格蛋白衣被组装和货物选择方面发挥着核心作用。在这里，我们通过一种受酵母内吞衔接蛋白 Sla1p 的 SHD2 结构域负调控的变体网格蛋白结合基序来描述 Sla1p 与网格蛋白的结合。SHD2 的晶体结构将该结构域鉴定为一个无菌α基序 (SAM) 结构域，并显示出寡聚化的倾向。通过共免疫沉淀，Sla1p 在体内与网格蛋白结合并自我聚集。破坏网格蛋白结合的网格蛋白结合基序突变以及阻碍自组装的基于结构的 SHD2 突变导致胞吞作用缺陷和 Sla1p 在胞吞作用部位组装的动态变化。这些结果定义了衔接蛋白负调控网格蛋白结合的一种新机制，并表明 SAM 结构域在网格蛋白介导的胞吞作用中发挥作用。
