Feliciano Daniel, Tolsma Thomas O, Farrell Kristen B, Aradi Al, Di Pietro Santiago M
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Traffic. 2015 Apr;16(4):379-97. doi: 10.1111/tra.12259. Epub 2015 Feb 24.
During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.
在网格蛋白介导的内吞作用（CME）过程中，肌动蛋白组装提供驱动力以促使囊泡内化。威斯科特-奥尔德里奇综合征蛋白（WASP）家族成员在刺激肌动蛋白组装方面发挥着重要作用。WASP家族蛋白包含一个结合球状肌动蛋白（G-肌动蛋白）的WH2基序和一个结合Arp2/3复合物的中央酸性基序，从而促进分支状肌动蛋白丝的形成。酵母WASP（Las17）是CME过程中促进Arp2/3依赖性肌动蛋白聚合的五个因子中作用最强的。有人提出，这种强大的活性可能是由Las17中一个假定的第二个G-肌动蛋白结合基序引起的。在此，我们描述了这种Las17 G-肌动蛋白结合基序（LGM）的体外和体内特性及其对一组保守精氨酸残基的依赖性。通过酵母双杂交系统、GST下拉实验、荧光偏振和芘-肌动蛋白聚合实验，我们表明LGM结合G-肌动蛋白，并且是体外正常Arp2/3介导的肌动蛋白聚合所必需的。活细胞荧光显微镜实验表明，LGM是CME过程中肌动蛋白聚合正常动态所必需的。此外，LGM对于在内吞作用的早期、中期和晚期募集的内吞机制成分的正常动态也是必需的，对于天然CME货物的最佳内吞作用也是如此。体外和体内实验均表明，与先前已知的Las17 G-肌动蛋白结合基序WH2相比，LGM的效力相对较低。这些结果在Las17中确立了第二个G-肌动蛋白结合基序，并推进了我们对CME过程中肌动蛋白组装机制的认识。
