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

Nature Structural & Molecular Biology volume 29, pages 339–347 (2022)Cite this article

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Abstract

Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.

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Fig. 1: Cooperative engagement of AP2 by muniscins and membrane mimetic.
Fig. 2: The primed conformation of AP2.
Fig. 3: Cargo-binding pockets are exposed in the primed AP2 complex.
Fig. 4: YxxΦ-cargo-binding releases AP2 from muniscins.
Fig. 5: YxxΦ-cargo converts primed AP2 complexes to the open conformation on membranes.
Fig. 6: Primed AP2 stimulates assembly of cargo-containing clusters on membranes.
Fig. 7: Model for adaptor checkpoint to catalyze cargo incorporation.

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Data availability

Cryo-EM data are deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMBD-24710, EMBD-24711, EMBD-24712, EMBD-24713 and EMBD-24714. For each deposition, the unsharpened map, sharpened map, unfiltered half maps and refinement mask were provided. Associated atomic coordinates are deposited in the PDB under accession numbers: 7RW8, 7RW9, 7RWA, 7RWB and 7RWX. The following atomic coordinates from the PDB were used in this study: 2VGL, 1BXX, 2JKR and 2XA7. Source data are provided with this paper.

Code availability

We used a customized build of Relion, which is available at https://github.com/Rick-Baker/relion-recenter.

References

  1. Zaremba, S. & Keen, J. H. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J. Cell Biol. 97, 1339–1347 (1983).

    Article  CAS  PubMed  Google Scholar 

  2. Pearse, B. M. & Robinson, M. S. Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 3, 1951–1957 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. & Owen, D. J. Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523–535 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Ohno, H. et al. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269, 1872–1875 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327–1332 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kelly, B. T. et al. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456, 976–979 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jackson, L. P. et al. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell 141, 1220–1229 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kelly, B. T. et al. Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch. Science 345, 459–463 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kovtun, O., Dickson, V. K., Kelly, B. T., Owen, D. J. & Briggs, J. A. G. Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles. Sci. Adv. 6, eaba8381 (2020).

  10. Schmid, E. M. & McMahon, H. T. Integrating molecular and network biology to decode endocytosis. Nature 448, 883–888 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Brach, T., Godlee, C., Moeller-Hansen, I., Boeke, D. & Kaksonen, M. The initiation of clathrin-mediated endocytosis is mechanistically highly flexible. Curr. Biol. 24, 548–554 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Höning, S. et al. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. Mol. Cell 18, 519–531 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Puthenveedu, M. A. & von Zastrow, M. Cargo regulates clathrin-coated pit dynamics. Cell 127, 113–124 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Loerke, D. et al. Cargo and dynamin regulate clathrin-coated pit maturation. PLoS Biol. 7, e57 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Cocucci, E., Aguet, F., Boulant, S. & Kirchhausen, T. The first five seconds in the life of a clathrin-coated pit. Cell 150, 495–507 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ma, L. et al. Transient Fcho1/2⋅ Eps15/R⋅ AP-2 nanoclusters prime the AP-2 clathrin adaptor for cargo binding. Dev. Cell 37, 428–443 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kadlecova, Z. et al. Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2. J. Cell Biol. 216, 167–179 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bhave, M. et al. Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics. Proc. Natl Acad. Sci. USA 117, 31591–31602 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Stimpson, H. E. M., Toret, C. P., Cheng, A. T., Pauly, B. S. & Drubin, D. G. Early-arriving Syp1p and Ede1p function in endocytic site placement and formation in budding yeast. Mol. Biol. Cell 20, 4640–4651 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Reider, A. et al. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation. EMBO J. 28, 3103–3116 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Henne, W. M. et al. FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 1281–1284 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Uezu, A. et al. SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15. J. Biol. Chem. 282, 26481–26489 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Mayers, J. R. et al. Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc. Natl Acad. Sci. USA 110, 11857–11862 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Traub, L. M. A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation. eLife 8, e41768 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Hollopeter, G. et al. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex. eLife 3, e03648 (2014).

    Article  PubMed Central  Google Scholar 

  26. Umasankar, P. K. et al. A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing. eLife 3, e04137 (2014).

    Article  PubMed Central  Google Scholar 

  27. Sochacki, K. A., Dickey, A. M., Strub, M.-P. & Taraska, J. W. Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells. Nat. Cell Biol. 19, 352–361 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Henne, W. M. et al. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature. Structure 15, 839–852 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Gaidarov, I. & Keen, J. H. Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. J. Cell Biol. 146, 755–764 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lehmann, M. et al. Nanoscale coupling of endocytic pit growth and stability. Sci. Adv. 5, eaax5775 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Matsui, W. & Kirchhausen, T. Stabilization of clathrin coats by the core of the clathrin-associated protein complex AP-2. Biochemistry 29, 10791–10798 (1990).

    Article  CAS  PubMed  Google Scholar 

  33. Aguilar, R. C., Ohno, H., Roche, K. W. & Bonifacino, J. S. Functional domain mapping of the clathrin-associated adaptor medium chains μ1 and μ2. J. Biol. Chem. 272, 27160–27166 (1997).

    Article  CAS  PubMed  Google Scholar 

  34. Beacham, G. M., Partlow, E. A., Lange, J. J. & Hollopeter, G. NECAPs are negative regulators of the AP2 clathrin adaptor complex. eLife 7, e32242 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Partlow, E. A. et al. A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex. eLife 8, e50003 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  36. El Alaoui, F. et al. Structural organization and dynamics of FCHo2 docking on membranes. eLife 11, e73156 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Ren, X., Farías, G. G., Canagarajah, B. J., Bonifacino, J. S. & Hurley, J. H. Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1. Cell 152, 755–767 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Jia, X. et al. Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1. eLife 3, e02362 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dacks, J. B. & Robinson, M. S. Outerwear through the ages: evolutionary cell biology of vesicle coats. Curr. Opin. Cell Biol. 47, 108–116 (2017).

    Article  CAS  PubMed  Google Scholar 

  40. Gopalakrishnan, G., Rouiller, I., Colman, D. R. & Lennox, R. B. Supported bilayers formed from different phospholipids on spherical silica substrates. Langmuir 25, 5455–5458 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Cannon, K. S., Woods, B. L., Crutchley, J. M. & Gladfelter, A. S. An amphipathic helix enables septins to sense micrometer-scale membrane curvature. J. Cell Biol. 218, 1128–1137 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  43. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Asarnow, D., Palovcak, E. & Cheng, Y. asarnow/pyem: UCSF pyem v0.5. Zenodo https://doi.org/10.5281/zenodo.3576630 (2019).

  49. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D. Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We acknowledge J. Peck and J. Strauss of the UNC Cryo-EM Core Facility for technical assistance in this project. We thank A. Gladfelter from UNC Chapel Hill and J. Chappie and G. Beacham from Cornell University for use of reagents and equipment. We thank R. Cerione, S. Emr, C. Adler and C. Fromme from Cornell University for thoughtful suggestions to improve the manuscript. Funding was provided by National Institutes of Health grant no. R01GM127548 (G.H.)

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Authors and Affiliations

  1. Department of Molecular Medicine, Cornell University, Ithaca, NY, USA

    Edward A. Partlow & Gunther Hollopeter

  2. Department of Biochemistry and Biophysics, University of North Carolina (UNC) Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Kevin S. Cannon & Richard W. Baker

  3. UNC Lineberger Comprehensive Cancer Center, University of North Carolina (UNC) Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Richard W. Baker

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  1. Edward A. Partlow
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Contributions

Conceptualization was done by E.A.P., G.H. and R.W.B. The methodology was devised by E.A.P., K.S.C., G.H. and R.W.B. The investigation was carried out by E.A.P., K.S.C., G.H. and R.W.B. The visualization was done by E.A.P., K.S.C. and R.W.B. Funding was acquired by G.H. and R.W.B. Project administration was done by G.H. Supervision was undertaken by G.H. and R.W.B. The original draft was written by E.A.P. Review and editing of the manuscript was done by E.A.P., K.S.C., G.H. and R.W.B.

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Correspondence to Gunther Hollopeter or Richard W. Baker.

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Nature Structural & Molecular Biology thanks Sandra Schmid, Reza Paraan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Supporting data for Fig. 1.

(a) Alignment of muniscin APA domains from four model organisms. Vertebrates (H. sapiens, D. rerio) contain two homologs of FCHo and the neuronal SGIP1. D. Melanogaster and C. elegans each contain a single muniscin. Black boxes: 100% identity, full color: 100% similarity, partial color: >75% similarity. (b) Confocal slices of AP2core bound to lipid-coated beads alone or pre-seeded with muniscin. Both proteins are added at 50 nM concentration. Scale bar 5 µm. FCHoBAR and FCHoBAR+APA images are also used in Fig. 1f. (c) Maximum intensity projections of AP2core (blue) and FCHoBAR+APA (green) bound to beads. Both proteins are added at 50 nM concentration. (d) Representative images of three assay conditions: AP2 alone, AP2 + FCHoBAR, and AP2 + FCHoBAR+APA. (e) Puncta formation of AP2core in the presence of soluble SGIPAPA at three concentrations. AP2 is held at 2 µM concentration. (f) Representative images of AP2 binding to SLBs +/- SGIPMP+APA, which includes the membrane phospholipid-binding domain (MP) and APA domain of SGIP. (g) Quantification of SLBs showing that AP2 binding is increased by the addition of 50 nM SGIPMP+APA. (h) Quantification of SLBs showing that the number of AP2 puncta per bead is increased by the addition of 50 nM SGIPMP+APA. (G-H) Data are mean +/- sd of triplicate measurements. ****P < 0.0001 for unpaired t-test (i) Representative pulldown of AP2 by either HT:SGIPAPA, HT:SGIPMP+APA, or HT:FCHo2BAR+APA in the presence of 5 µM heparin. HT: HaloTag. (j) Representative full SDS-PAGE gels of the protease sensitivity experiments quantified in Fig. 1g. Data for graphs in g,h are available as source data.

Source data

Extended Data Fig. 2 Data collection, processing, and model building for the AP2core-heparin complex.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right) (b) 2D class averages of the ‘clean’ dataset that was used for ab initio model generation. Two classes were found for further processing. (c) Final cryo-EM map of the ‘closed’ AP2-heparin complex colored by local resolution. Standard FSC plots (d) and directional FSC plots (e) are shown. (f) Final cryo-EM map of the ‘bowl’ AP2-heparin complex colored by local resolution. Note the µ2-CTD has been ejected from the complex and is not resolved in this map. Standard FSC plots (g) and directional FSC plots (h) are shown. (i) Pseudo-difference density map for the AP2 bowl complex made in Chimera and colored red to show density that cannot be accounted for by AP2. A region of low-resolution density is seen on the surface of α, corresponding to a known PIP2 binding site. Additional density is found at the α/σ2 interface. (j) Same analysis as done in (I), but for the closed AP2-heparin complex. (k) The N-terminus of ß2 packs into the [D/E]xxL[L/I] binding pocket on σ2 (yellow) (l) The structures of the closed AP2 (2VGL.pdb) and [D/E]xxL[L/I]-bound unlatched AP2 (2JKR.pdb) are shown aligned to the model for AP2-heparin. The closed heparin structure shows an intermediate conformation between the closed apo and [D/E]xxL[L/I]-cargo bound states.

Extended Data Fig. 3 Cryo-EM structure determination of AP2-heparin-YxxΦ-cargo.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right) (b) Representative cryo-EM micrograph and particle picking workflow. (c) 2D class averages (middle, right) of the ‘clean’ dataset that was used for ab initio model generation (left). (d) Final cryo-EM map of the AP2-heparin-YxxΦ complex colored by local resolution. (e) Gold standard FSC plot. (f) Particle distribution plot for 3D refinement. (g) Directional FSC plot. (h) The sharpened map is shown with potential heparin density colored red. Two copies of AP2-YxxΦ are shown docked into the cryo-EM map (right). (i) The refined model (grey) is overlaid with the ‘open’ AP2-YxxΦ structure (2xa7.pdb).

Extended Data Fig. 4 Data collection and processing workflow for the AP2core-heparin-SGIPAPA complex.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right). (b) 2D class averages of the ‘clean’ dataset that was used for ab initio model generation. Two classes were found for further processing. (c) Final cryo-EM map of the dimeric AP2core-heparin-SGIPAPA complex colored by local resolution Standard FSC plots (d) and directional FSC plots (e) are shown. (f) FSC plot for the monomer class 3D refinement. (g) Refined monomer map is shown with opaque and transparent surface with a docked model of AP2 that was manually segmented and re-arranged in UCSF Chimera, showing a preliminary assignment of density to AP2 subunits. (h) The AP2core-heparin-SGIPAPA dimeric cryo-EM map is shown (left) and with a transparent surface and docked molecular models (middle). A pseudo difference map was generated in Chimera and is shown colored pink. (i) Density along the dimeric interface is shown. This density is lined by known PIP2 binding residues (colored red), so we assign this density to heparin.

Extended Data Fig. 5 Assigning the conformation of the µ2-CTD in the dimeric AP2core-heparin-SGIPAPA structure.

(a) The AP2core-heparin-SGIPAPA cryo-EM map (left) contains a symmetry-related dimer of two complexes assembled on the same heparin molecule. The resolution of our structure in combination with the flexibility of the linker connecting the µ2-NTD and the µ2-CTD precludes us from unambiguously identifying which ‘bowl’ contributes which µ2-CTD, resulting in 2 candidate monomers (center and right). (b) Surface representation of candidate monomers with PIP2-binding residues shaded red and YxxΦ cargo-binding residues shaded orange. (c) Surface representation of candidate monomers along with surface representations of the ‘bowl’ and µ-CTD for each. Magenta represents buried surface area, totaling 499 Å2 for candidate 1 (left), and 199 Å2 for candidate 2 (right).

Extended Data Fig. 6 AP2core-heparin-SGIPAPA symmetry expansion and SGIPAPA model building.

(a) Density that is not accounted for by AP2 subunits in the AP2core-heparin-SGIPAPA structure is shown in pink. Density on the surface of β2 we assign to SGIPAPA as it is not observed in AP2-heparin structures and all residues at the α and β2 termini are accounted for. (b) Masks used for partial signal subtraction and manual symmetry expansion. (c) AP2-heparin-SGIPAPA ‘monomer’ refinement using signal subtracted particles. A map filtered by local resolution is shown colored by local resolution (left). The sharpened map is shown (grey) along with the FSC plot. (d) The poly-ALA model of SGIPAPA is shown in the density between β2 helices 25 and 27.

Extended Data Fig. 7 AP2 pull-down assays in the presence of cargo peptide.

(a) Representative pulldown of AP2 by HT:SGIPAPA in the presence of 5 µM heparin and either YxxΦ or ExxxLL cargo peptide. HT: HaloTag. (b) Representative pulldown of AP2 by either HT:SGIPAPA or HT:FCHo2BAR+APA in the presence of 5 µM heparin and YxxΦ-cargo peptide. HT: HaloTag. Uncropped image for b is available as source data.

Source data

Extended Data Fig. 8 Engineered disulfide crosslinks to probe AP2 conformation.

(a) Cartoon representation of the closed, primed, and open structures showing the location and Cß-Cß distances of all three engineered disulfides. (b) Representative uncropped Western blots for µ2 showing the primed-conformation disulfide experiment quantified in Fig. 3e.

Extended Data Fig. 9 Uncropped gels from disulfide crosslinking experiments.

Gels showing the technical replicates quantified in Fig. 5 are shown for the primed-specific disulfide (a), the closed-specific disulfide (b), and the open-specific disulfide (c). All gels are ordered as shown in (A).

Extended Data Fig. 10 Examples of three-color fluorescence imaging.

(a) Five lipid-coated beads are shown in three separate imaging channels and merged. Beads contain 2% YxxΦ-cargo and were incubated with 1 µM FCHoBAR and 1 µM AP2core. (b) Five example beads are shown as in (A), but were incubated with 1 µM FCHoBAR+APA. (c) and (d) show the same experiment in (A) and (B), but using SGPMP+APA. (e) Quantification of beads containing YxxΦ-cargo puncta with or without SGIPMP+APA Data are mean +/- sd of duplicate measurements. ****P < 0.0001 for unpaired t-test (f) Quantification of the AP2 to lipid ratio in cargo puncta with or without SGIPMP+APA. Data are mean +/- sd of triplicate measurements. ****P < 0.0001 for unpaired t-test. Data for graphs in e,f are available as source data. Scale bar: 5 µm.

Source data

Supplementary information

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 1

Numerical data for graphs.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 4

Numerical data for graphs.

Source Data Fig. 5

Numerical data for graphs.

Source Data Fig. 6

Numerical data for graphs.

Source Data Extended Data Fig. 1

Numerical data for graphs.

Source Data Extended Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 10

Numerical data for graphs.

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Partlow, E.A., Cannon, K.S., Hollopeter, G. et al. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization. Nat Struct Mol Biol 29, 339–347 (2022). https://doi.org/10.1038/s41594-022-00749-z

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