A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

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Abstract

Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.

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All data generated or analysed during this study are included in the manuscript and supporting files. Key plasmids will be made available via Addgene.

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Linton M Traub

Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States

For correspondence
traub@pitt.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1303-0298

Funding

National Institute of General Medical Sciences (R01GM106963)

Linton M Traub

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.