Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests that adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea, we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 domain point mutations that disrupted NPFxD binding caused a proportional reduction in Sla1–GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 domain point mutation and deletion of the C-terminal LxxQxTG repeat (SR) region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1–GFP recruitment to the plasma membrane. These data suggest that multiple interactions are needed for recruitment of Sla1 to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain, which binds ubiquitin, and the SHD1 domain displayed broad surface localization, suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitylated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region might regulate the Sla1–cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.

质膜蛋白的内吞作用是通过它们与衔接蛋白的相互作用介导的。相反，新出现的证据表明，衔接蛋白招募到质膜可能取决于与内吞货物的结合。为了测试这个想法，我们分析了酵母接头蛋白 Sla1，它通过 Sla1 SHD1 结构域结合带有内吞信号 NPFxD 的膜蛋白。一致的是，破坏 NPFxD 结合的 SHD1 结构域点突变导致 Sla1-GFP 招募到内吞位点的比例减少。此外，同时 SHD1 结构域点突变和连接 Sla1 与外壳蛋白 Pan1 和 End3 的 C 端 LxxQxTG 重复 (SR) 区域的缺失导致 Sla1-GFP 募集到质膜的完全丧失。这些数据表明 Sla1 募集到膜上需要多种相互作用。有趣的是，Sla1 片段仅包含第三个 SH3 结构域（与泛素结合），而 SHD1 结构域显示出广泛的表面定位，表明质膜募集是通过与含有 NPFxD 和泛素化质膜蛋白的相互作用介导的。我们的结果还表明，与 SR 区域相邻的 Sla1 NPF 基序可能调节 Sla1-货物相互作用，从机制上将 Sla1 货物结合与内吞位点招募联系起来。