Handling Editor: Mahak Sharma
ABSTRACT
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.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: T.O.T., S.M.D.P.; Methodology: T.O.T., H.P.F., D.M.O.; Validation: T.O.T.; Formal analysis: T.O.T., H.P.F., S.M.D.P.; Investigation: T.O.T., H.P.F., D.M.O.; Data curation: T.O.T., H.P.F., S.M.D.P.; Writing - original draft: T.O.T.; Writing - review & editing: T.O.T., S.M.D.P.; Visualization: T.O.T., S.M.D.P.; Supervision: S.M.D.P.; Project administration: S.M.D.P.; Funding acquisition: S.M.D.P.
Funding
This work was supported by National Science Foundation grant MCB-1616775 and National Institutes of Health grant GM-125619 to S.M.D.P. An American Heart Association predoctoral fellowship provided support to T.O.T. Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at https://jcs.biologists.org/lookup/doi/10.1242/jcs.247684.supplemental
- Received April 17, 2020.
- Accepted August 27, 2020.
- © 2020. Published by The Company of Biologists Ltd
Log in using your username and password
Log in through your institution
Pay Per Article - You may access this article (from the computer you are currently using) for 1 day for US$30.00 .
Regain Access - You can regain access to a recent Pay per Article purchase if your access period has not yet expired.