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TitleFCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch.
Journal, issue, pagesSci Adv, Vol. 8, Issue 17, Page eabn2018, Year 2022
Publish dateApr 29, 2022
AuthorsNathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / Sally R Gray / Kun Qu / Philip R Evans / Marco Fritzsche / Filip Sroubek / Stefan Höning / John A G Briggs / Bernard T Kelly / David J Owen / Linton M Traub /
PubMed AbstractClathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane- ...Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.
External linksSci Adv / PubMed:35486718 / PubMed Central
MethodsEM (single particle) / EM (tomography) / EM (subtomogram averaging) / X-ray diffraction
Resolution1.41 - 12.0 Å
Structure data

EMDB-14517, PDB-7z5c:
Chimera of AP2 with FCHO2 linker domain as a fusion on Cmu2 subunit
Method: EM (single particle) / Resolution: 4.16 Å

EMDB-14518: Chimaera of AP2 with FCHO2 linker domain, N1-N2 enriched population
Method: EM (single particle) / Resolution: 6.7 Å

EMDB-14525: AP2 adaptor protein recruited on the membrane in the presence of FCHO2 linker
Method: EM (tomography) / Resolution: 9.9 Å

EMDB-14526: AP2 on the membrane without cargo peptide
Method: EM (subtomogram averaging) / Resolution: 12.0 Å

PDB-7ofp:
Apo Structure of Mu2 Adaptin Subunit (Ap50) Of AP2 Clathrin Adaptor
Method: X-RAY DIFFRACTION / Resolution: 1.92 Å

PDB-7og1:
AP2 clathrin adaptor core in complex with cargo peptide and FCHO2
Method: X-RAY DIFFRACTION / Resolution: 3.25 Å

PDB-7ohi:
FCHO1-peptide-AP2 alpha ear complex
Method: X-RAY DIFFRACTION / Resolution: 1.41 Å

PDB-7oho:
Crystal structure of AP2 FCHO2 chimera
Method: X-RAY DIFFRACTION / Resolution: 2.88 Å

PDB-7ohz:
Crystal structure of AP2 Mu2 - FCHO2 chimera (His6-tagged)
Method: X-RAY DIFFRACTION / Resolution: 2.27 Å

PDB-7oi5:
Crystal structure of AP2 Mu2 - FCHO2 chimera (GST cleaved)
Method: X-RAY DIFFRACTION / Resolution: 2.61 Å

PDB-7oiq:
Crystal structure of AP2 Mu2 in complex with FCHO2 WxxPhi motif (C2 crystal form)
Method: X-RAY DIFFRACTION / Resolution: 1.85 Å

PDB-7oit:
Crystal structure of AP2 Mu2 in complex with FCHO2 WxxPhi motif (P3221 crystal form)
Method: X-RAY DIFFRACTION / Resolution: 1.65 Å

Chemicals

ChemComp-GOL:
GLYCEROL

ChemComp-CIT:
CITRIC ACID

ChemComp-HOH:
WATER

ChemComp-SO4:
SULFATE ION

ChemComp-MES:
2-(N-MORPHOLINO)-ETHANESULFONIC ACID / pH buffer*YM

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE

Source
  • Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
  • rattus norvegicus (Norway rat)
  • homo sapiens (human)
  • mus musculus (house mouse)
KeywordsENDOCYTOSIS / clathrin-mediated endocytosis (CME) / YxxPhi motif / Pi(4 / 5)P2 / protein recycling / plasma membrane / PROTEIN TRANSPORT / adaptor complex / AP2 / trafficking / clathrin / CCP

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