Burston Helen E, Maldonado-Báez Lymarie, Davey Michael, Montpetit Benjamen, Schluter Cayetana, Wendland Beverly, Conibear Elizabeth
Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver V5Z 4H4, British Columbia, Canada.
J Cell Biol. 2009 Jun 15;185(6):1097-110. doi: 10.1083/jcb.200811116. Epub 2009 Jun 8.
Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.
质膜上受体的内吞作用受一种复杂机制控制，该机制包括网格蛋白、衔接蛋白和肌动蛋白调节因子。这些蛋白中的许多在酵母中是保守的，但缺乏可观察到的突变表型，这表明酵母内吞作用可能受不同的调控机制影响。在这里，我们使用了一种全基因组定量筛选方法系统地确定了内化所需的基因，该方法监测酵母囊泡相关膜蛋白（VAMP）/突触融合蛋白同源物Snc1的定位。遗传相互作用图谱被用于将这些基因归入包含已知和新型内吞调节因子的功能模块，并通过基于阵列的比较分析评估货物选择性。我们证明，网格蛋白和酵母AP180网格蛋白衔接蛋白在Snc1内化过程中具有货物特异性作用。我们还鉴定出低染料结合蛋白17（LDB17）是内吞机制的一种新型保守成分。Ldb17在肌动蛋白聚合之前被招募到皮质肌动蛋白斑块，并调节正常的包被动态和肌动蛋白组装。我们的研究结果突出了保守机制，并揭示了内吞内化的新机制。
