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.
Highlights
Clathrin-mediated endocytosis is an evolutionarily ancient mode of directed transport of selected macromolecules into the eukaryotic cell interior
Even in the absence of EGF receptor activation, epidermal growth factor receptor pathway substrate 15 (Eps15) localizes to the vast majority of cell surface clathrin-coated structures at steady state, is positioned predominantly at the margins of assembling lattices (Sochacki et al, 2017; Tebar et al, 1996; Henne et al, 2010; Edeling et al, 2006), and is among the first assembled coat proteins encountered by an activated G protein-coupled
The FCHO1-corrected phenotype is starkly different from the untransfected clone #40 cells, and closely mirrors the results seen in clone #64/1.E cells (Umasankar et al, 2014; Sager et al, 1984), where we have shown this is not due to the phospholipid-binding N-terminal EFC domain (Umasankar et al, 2014)
Summary
Clathrin-mediated endocytosis is an evolutionarily ancient mode of directed transport of selected macromolecules into the eukaryotic cell interior. Even in the absence of EGF receptor activation, Eps localizes to the vast majority of cell surface clathrin-coated structures at steady state (van Delft et al, 1997a; Benmerah et al, 1999; Tebar et al, 1996), is positioned predominantly at the margins of assembling lattices (Sochacki et al, 2017; Tebar et al, 1996; Henne et al, 2010; Edeling et al, 2006), and is among the first assembled coat proteins encountered by an activated G protein-coupled
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