Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests 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 point mutations that disrupt NPFxD binding caused a proportional reduction of Sla1-GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 point mutation and deletion of the SR region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1-GFP recruitment to the plasma membrane. The data suggests multiple interactions are needed for recruitment to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain – which binds ubiquitin – and SHD1, displayed broad surface localization suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitinated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region may regulate the Sla1-cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.
Cargo-Mediated Recruitment of the Endocytic Adaptor Protein Sla1
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Thomas O. Tolsma, Hallie P. Febvre, Deanna M. Olson, Santiago M. Di Pietro; Cargo-Mediated Recruitment of the Endocytic Adaptor Protein Sla1. J Cell Sci 2020; jcs.247684. doi: https://doi.org/10.1242/jcs.247684
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