Article ; Online: A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation.
2019 Volume 8
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. ...
| 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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| MeSH term(s) | Adaptor Protein Complex 2/metabolism ; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Amino Acid Sequence ; Cell Membrane/metabolism ; Clathrin/chemistry ; Clathrin/genetics ; Clathrin/metabolism ; Fatty Acid-Binding Proteins/metabolism ; Gene Editing ; HeLa Cells ; Humans ; Membrane Proteins/metabolism ; Protein Binding ; Protein Domains ; Protein Interaction Domains and Motifs ; Recombinant Fusion Proteins/metabolism ; Recombinant Proteins ; Sequence Alignment ; Single-Domain Antibodies/chemistry ; Single-Domain Antibodies/metabolism |
| Chemical Substances | Adaptor Protein Complex 2 ; Adaptor Proteins, Signal Transducing ; Clathrin ; EPS15 protein, human ; FCHO1 protein, human ; FCHO2 protein, human ; Fatty Acid-Binding Proteins ; Membrane Proteins ; Recombinant Fusion Proteins ; Recombinant Proteins ; Single-Domain Antibodies |
| Language | English |
| Publishing date | 2019-04-30 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, N.I.H., Extramural |
| ZDB-ID | 2687154-3 |
| ISSN | 2050-084X ; 2050-084X |
| ISSN (online) | 2050-084X |
| ISSN | 2050-084X |
| DOI | 10.7554/eLife.41768 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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