A Nanobody-based Molecular Toolkit Provides New Mechanistic Insight into Clathrin-coat Initiation

Overview

Journal Elife

Specialty Biology

Date 2019 May 1

PMID 31038455

Citations 13

Authors

Linton M Traub

Affiliations

Soon will be listed here.

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.

Citing Articles

The conserved protein adaptors CALM/AP180 and FCHo1/2 cooperatively recruit Eps15 to promote the initiation of clathrin-mediated endocytosis in yeast.

Sun Y, Yeam A, Kuo J, Iwamoto Y, Hu G, Drubin D PLoS Biol. 2024; 22(9):e3002833.

PMID: 39316607 PMC: 11451990. DOI: 10.1371/journal.pbio.3002833.

Generation of nanoscopic membrane curvature for membrane trafficking.

Kozlov M, Taraska J Nat Rev Mol Cell Biol. 2022; 24(1):63-78.

PMID: 35918535 DOI: 10.1038/s41580-022-00511-9.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch.

Zaccai N, Kadlecova Z, Dickson V, Korobchevskaya K, Kamenicky J, Kovtun O Sci Adv. 2022; 8(17):eabn2018.

PMID: 35486718 PMC: 9054013. DOI: 10.1126/sciadv.abn2018.

Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.

Partlow E, Cannon K, Hollopeter G, Baker R Nat Struct Mol Biol. 2022; 29(4):339-347.

PMID: 35347313 PMC: 10116491. DOI: 10.1038/s41594-022-00749-z.

Synaptic AP2 CCV life cycle regulation by the Eps15, ITSN1, Sgip1/AP2, synaptojanin1 interactome.

Mishra R, Sengul G, Candiello E, Schu P Sci Rep. 2021; 11(1):8007.

PMID: 33850201 PMC: 8044098. DOI: 10.1038/s41598-021-87591-3.

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