Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.
Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.
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DOI：
10.1371/journal.pone.0136732
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Ayscough KR
中科院分区：
文献类型：
--
作者：
Urbanek AN;Allwood EG;Smith AP;Booth WI;Ayscough KR
在酿酒酵母的内吞作用过程中，肌动蛋白聚合被认为为内陷提供驱动力，以抵抗膨压的影响。在之前的研究中，Ysc84 被证明可以通过保守的 N 末端结构域结合肌动蛋白。然而，全长 Ysc84 仅当其 C 端 SH3 结构域也与酵母 WASP 同源物 Las17 结合时才能结合肌动蛋白。活细胞成像显示，Ysc84 在 Las17/WASP 之后但在其他已知的肌动蛋白结合蛋白之前定位于内吞位点，这表明它可能在膜内陷的早期阶段发挥作用。虽然其他生物体中存在 Ysc84 的同源物，包括其人类同源物 SH3yl-1，但人们对其与肌动蛋白相互作用的模式或这种相互作用如何影响肌动蛋白丝动力学知之甚少。在这里，我们鉴定了参与 Ysc84 肌动蛋白和脂质结合的关键残基，并证明其肌动蛋白结合活性受到 PI(4,5)P2 的负调节。在体内表征了脂质或肌动蛋白结合相互作用缺陷的 Ysc84 突变体。 Ysc84 通过其 C 端 SH3 结构域结合 Las17 或通过 N 端结构域结合肌动蛋白和脂质的能力都被证明对于拯救需要 YSC84 表达的菌株中的温度敏感生长至关重要。带有荧光标记的内吞报告蛋白的菌株的活细胞成像揭示了突变体的不同表型，表明这些相互作用对于调节内吞作用关键阶段的重要性。
During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis.