Sun Yidi, Kaksonen Marko, Madden David T, Schekman Randy, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Mol Biol Cell. 2005 Feb;16(2):717-30. doi: 10.1091/mbc.e04-08-0740. Epub 2004 Dec 1.
A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.
多项研究表明脂质磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）参与内吞内化过程，但这种脂质如何介导其作用尚不清楚。AP180 N端同源（ANTH）结构域是一种PtdIns(4,5)P2结合模块，存在于几种参与受体介导内吞作用的蛋白质中。酵母Sla2p就是这样一种蛋白质，它是一种高度保守的肌动蛋白结合蛋白，对肌动蛋白组织和内吞内化至关重要。为了更好地理解PtdIns(4,5)P2结合如何调节肌动蛋白依赖性内吞作用，我们研究了Sla2p的ANTH结构域的功能。脂质体结合试验表明，Sla2p通过其ANTH结构域特异性结合PtdIns(4,5)P2，并确定了这种相互作用所需的特定赖氨酸残基。缺乏PtdIns(4,5)P2结合的Sla2p突变体在细胞生长、肌动蛋白组织和内吞内化方面表现出明显缺陷。这些缺陷可以通过提高体内PtdIns(4,5)P2水平来挽救。令人惊讶的是，在PtdIns(4,5)P2结合方面有缺陷的突变体Sla2p与细胞皮质的内吞机制定位在一起，这表明ANTH-PtdIns(4,5)P2相互作用对于这种关联不是必需的。相比之下，多色实时荧光显微镜和颗粒追踪分析表明，在胞吞内化过程中需要PtdIns(4,5)P2结合。这些结果表明，Sla2p的ANTH结构域与PtdIns(4,5)P2的相互作用在调节肌动蛋白依赖性内吞内化动力学中起关键作用。
