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• W2097138052 abstract "•CALM loss increases size and frequency of early endocytic clathrin-coated structures•Depletion of CALM slows endocytic clathrin-coated pit maturation and endocytic rate•CALM possesses an N-terminal, membrane-curvature-sensing/driving amphipathic helix•Clathrin-coated pit maturation is regulated by CALM’s N-terminal amphipathic helix The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. During clathrin-mediated endocytosis (CME), clathrin-coated pits (CCPs) are most frequently initiated by the formation of a small patch of the heterotetrameric AP2 clathrin adaptor complex and clathrin at sites of high PtdIns4,5P2 concentration (Cocucci et al., 2012Cocucci E. Aguet F. Boulant S. Kirchhausen T. The first five seconds in the life of a clathrin-coated pit.Cell. 2012; 150: 495-507Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, Traub, 2011Traub L.M. Regarding the amazing choreography of clathrin coats.PLoS Biol. 2011; 9: e1001037Crossref PubMed Scopus (37) Google Scholar). If not aborted due to a lack of sufficient PtdIns4,5P2 and/or cargo (Loerke et al., 2009Loerke D. Mettlen M. Yarar D. Jaqaman K. Jaqaman H. Danuser G. Schmid S.L. Cargo and dynamin regulate clathrin-coated pit maturation.PLoS Biol. 2009; 7: e57Crossref PubMed Scopus (156) Google Scholar), local membrane curvature steadily increases as more clathrin and a variety of additional clathrin adaptors are recruited until a bulbous membrane structure of ∼80–100 nm diameter is formed on top of a membrane stalk that can undergo scission to generate a clathrin-coated vesicle (CCV) (Traub, 2011Traub L.M. Regarding the amazing choreography of clathrin coats.PLoS Biol. 2011; 9: e1001037Crossref PubMed Scopus (37) Google Scholar). The most abundant clathrin adaptors in endocytic CCVs isolated from tissue culture cells are CALM (clathrin assembly lymphoid myeloid leukemia protein) and AP2, each of which account for 30%–35% of the adaptors in a CCV (Blondeau et al., 2004Blondeau F. Ritter B. Allaire P.D. Wasiak S. Girard M. Hussain N.K. Angers A. Legendre-Guillemin V. Roy L. Boismenu D. et al.Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.Proc. Natl. Acad. Sci. USA. 2004; 101: 3833-3838Crossref PubMed Scopus (246) Google Scholar, Borner et al., 2012Borner G.H.H. Antrobus R. Hirst J. Bhumbra G.S. Kozik P. Jackson L.P. Sahlender D.A. Robinson M.S. Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles.J. Cell Biol. 2012; 197: 141-160Crossref PubMed Scopus (116) Google Scholar). AP2 binds to and sorts general, often large, transmembrane cargo such as the transferrin receptor (TfR) into CCVs. CALM binds to and sorts the small R-SNAREs VAMPs 2, 3, and 8 (Koo et al., 2011Koo S.J. Markovic S. Puchkov D. Mahrenholz C.C. Beceren-Braun F. Maritzen T. Dernedde J. Volkmer R. Oschkinat H. Haucke V. SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses.Proc. Natl. Acad. Sci. USA. 2011; 108: 13540-13545Crossref PubMed Scopus (99) Google Scholar, Miller et al., 2011Miller S.E. Sahlender D.A. Graham S.C. Höning S. Robinson M.S. Peden A.A. Owen D.J. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.Cell. 2011; 147: 1118-1131Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). CALM and its neuronal specific homolog AP180, possess large, natively unstructured, C-terminal tails containing clathrin, AP2 α- and β2-appendage binding sites (Ford et al., 2001Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.Science. 2001; 291: 1051-1055Crossref PubMed Scopus (602) Google Scholar, Tebar et al., 1999Tebar F. Bohlander S.K. Sorkin A. Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic.Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (240) Google Scholar, Traub, 2011Traub L.M. Regarding the amazing choreography of clathrin coats.PLoS Biol. 2011; 9: e1001037Crossref PubMed Scopus (37) Google Scholar), and a membrane-proximal, PtdIns4,5P2-binding N-terminal ANTH stacked-helical domain, defined as residues 19–289 (Ford et al., 2001Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.Science. 2001; 291: 1051-1055Crossref PubMed Scopus (602) Google Scholar). Several studies have shown that depletion of CALM in tissue culture cells and vertebrate neurons or of its orthologs in D. melanogaster and C. elegans causes an increase in CCP/CCV size and probably also a decrease in their uniformity of shape (Bao et al., 2005Bao H. Daniels R.W. MacLeod G.T. Charlton M.P. Atwood H.L. Zhang B. AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis.J. Neurophysiol. 2005; 94: 1888-1903Crossref PubMed Scopus (66) Google Scholar, Meyerholz et al., 2005Meyerholz A. Hinrichsen L. Groos S. Esk P.C. Brandes G. Ungewickell E.J. Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.Traffic. 2005; 6: 1225-1234Crossref PubMed Scopus (114) Google Scholar, Nonet et al., 1999Nonet M.L. Holgado A.M. Brewer F. Serpe C.J. Norbeck B.A. Holleran J. Wei L. Hartwieg E. Jorgensen E.M. Alfonso A. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles.Mol. Biol. Cell. 1999; 10: 2343-2360Crossref PubMed Scopus (227) Google Scholar, Petralia et al., 2013Petralia R.S. Wang Y.X. Indig F.E. Bushlin I. Wu F. Mattson M.P. Yao P.J. Reduction of AP180 and CALM produces defects in synaptic vesicle size and density.Neuromolecular Med. 2013; 15: 49-60Crossref PubMed Scopus (23) Google Scholar, Zhang et al., 1998Zhang B. Koh Y.H. Beckstead R.B. Budnik V. Ganetzky B. Bellen H.J. Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis.Neuron. 1998; 21: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). These morphological alterations most likely result from changes in membrane curvature/sculpting. However, structural investigations had shown that CALM possessed neither a BAR domain nor an amphipathic helix that could influence membrane curvature, which was consistent with CALM apparently not affecting membrane curvature in biophysical assays (Boucrot et al., 2012Boucrot E. Pick A. Çamdere G. Liska N. Evergren E. McMahon H.T. Kozlov M.M. Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains.Cell. 2012; 149: 124-136Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, Ford et al., 2002Ford M.G.J. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J.K. Evans P.R. McMahon H.T. Curvature of clathrin-coated pits driven by epsin.Nature. 2002; 419: 361-366Crossref PubMed Scopus (793) Google Scholar, Stahelin et al., 2003Stahelin R.V. Long F. Peter B.J. Murray D. De Camilli P. McMahon H.T. Cho W. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains.J. Biol. Chem. 2003; 278: 28993-28999Crossref PubMed Scopus (147) Google Scholar). In an attempt to reconcile these contradictory observations of a key cellular process, we set out to determine whether, and if so, how CALM could directly affect CCP/CCV size and thus the cargo-carrying capacity because these are two key features in understanding the mechanism of endocytic CCP/CCV formation. Here we show that CALM possesses an N-terminal amphipathic helix that regulates CCP/CCV size and maturation and hence endocytic rate. Endogenous CALM was depleted by siRNA and subsequent quantitated ultrastructural studies by electron microscopy (EM) and stimulated-emission depletion (STED) microscopy showed that CALM depletion results in substantially enlarged CCPs and CCVs compared to that of control cells (Figures 1, S1A, S1C, and S1E; see also Meyerholz et al., 2005Meyerholz A. Hinrichsen L. Groos S. Esk P.C. Brandes G. Ungewickell E.J. Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.Traffic. 2005; 6: 1225-1234Crossref PubMed Scopus (114) Google Scholar), but no discernable change in the number of clathrin-coated structures (Figures 1B and S1A). The CCPs/CCVs in control cells have an average diameter of ∼90 nm (Heuser, 1980Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts.J. Cell Biol. 1980; 84: 560-583Crossref PubMed Scopus (370) Google Scholar). This value increased by ∼2-fold upon depletion of CALM producing an ∼4-fold increase in surface area, hence an ∼8-fold increase in volume (Figures 1, S1C, and S1E). Importantly, our quantitation of CCP/CCV profiles of EM micrographs showed that “necked” CCPs/“closed” CCVs were the predominant fraction in control cells (∼70%), whereas the shallow “open” CCPs accounted for only ∼30% (Figures 1A and S1D). This was reversed in CALM-depleted cells, strongly suggestive of an alteration in the efficiency of CCP/CCV maturation, likely resulting from a reduced ability to curve/sculpt the plasma membrane (Figures 1A and S1D). In agreement with this, total internal reflection fluorescence (TIRF) microscopy revealed that the time taken to proceed from initiation of a CCP to its scission approximately doubles when CALM is depleted (Figures 2A and 2B ).Figure 2CALM Affects CCV Maturation and Possesses an Amphipathic Helix that Contributes to Membrane BindingShow full caption(A) The time between first detection of a CCP and the first detected scission event at that CCP (Δts) was significantly extended in cells treated with CALM siRNA (control cells, Δt = 59 s, SEM = 3 s, 688 events, five cells; CALM siRNA, Δt = 100 s, SEM = 4 s, 590 events, five cells).(B) An example CCP and scission event from a control cell. Images were acquired using TIRF microscopy and the pulsed pH protocol to detect single scission events. Images acquired at pH 7 (upper, Tf pH7) show a cluster of Transferrin-phluorin (Tf-phl) at a CCP. A scission event was detected when Tf-phl at the cluster became insulated from the externally imposed pH change (middle, Tf pH5, t = 0 s). Simultaneous detection of μ2-mCherry showed this CCP formed ∼62 s before the detected scission event (lower, arrow in μ2-mCherry graph). The quantified fluorescence changes for this example CCP are plotted (graphs).(C) Montages of example clathrin-coated pit nucleation events imaged using TIRF microscopy. The purple arrow indicates t = 0 s, the first frame in which the CALM-GFP (green) object was detected using automated tracking.(D) Average CALM-GFP and μ2-mCherry (red) fluorescence traces of nucleating CCPs aligned to the first frame of detection of CALM-GFP (green). The average traces were normalized between the average of the first three fluorescence values and the peak fluorescence value for CALM and μ2, respectively. The fluorescence traces have a characteristic sigmoidal shape.(E) Montages of example clathrin-coated pit budding events. The blue arrow indicates t = 0 s, the moment of maximum decrease in CALM-GFP fluorescence that corresponds to vesicle scission.(F) Average CALM-GFP and μ2-mCherry at budding clathrin-coated pits. The average fluorescence traces were normalized to the average of the first three and last three fluorescence values for CALM and μ2, respectively. The μ2-mCherry fluorescence starts to decrease ∼15 s before the CALM-GFP signal (black arrow).(G) Heliquest server (http://heliquest.ipmc.cnrs.fr) helical wheel shows the orientation of the key hydrophobic residues (in yellow) and predicts the N terminus of CALM is an amphipathic helix with curvature-sensing properties. The hydrophobic moment points up toward the membrane and polar but mainly uncharged side chains are disposed laterally along where the surface the membrane would be. The negative charge points down away from the membrane.(H) Liposome-based SPR sensorgrams showing binding of CALM ANTH-WT, CALM ANTH(ΔH0), CALM ANTH(H0mut), and CALM ANTH(PIP−) to PtdIns4,5P2-containing liposomes at a protein concentration of 6 μM.(I) KD values for the binding of CALM ANTH domains, determined by liposome-based SPR (mean and SD of four independent measurements). Representative sensorgrams used for the measurements are shown in Figure S2A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The time between first detection of a CCP and the first detected scission event at that CCP (Δts) was significantly extended in cells treated with CALM siRNA (control cells, Δt = 59 s, SEM = 3 s, 688 events, five cells; CALM siRNA, Δt = 100 s, SEM = 4 s, 590 events, five cells). (B) An example CCP and scission event from a control cell. Images were acquired using TIRF microscopy and the pulsed pH protocol to detect single scission events. Images acquired at pH 7 (upper, Tf pH7) show a cluster of Transferrin-phluorin (Tf-phl) at a CCP. A scission event was detected when Tf-phl at the cluster became insulated from the externally imposed pH change (middle, Tf pH5, t = 0 s). Simultaneous detection of μ2-mCherry showed this CCP formed ∼62 s before the detected scission event (lower, arrow in μ2-mCherry graph). The quantified fluorescence changes for this example CCP are plotted (graphs). (C) Montages of example clathrin-coated pit nucleation events imaged using TIRF microscopy. The purple arrow indicates t = 0 s, the first frame in which the CALM-GFP (green) object was detected using automated tracking. (D) Average CALM-GFP and μ2-mCherry (red) fluorescence traces of nucleating CCPs aligned to the first frame of detection of CALM-GFP (green). The average traces were normalized between the average of the first three fluorescence values and the peak fluorescence value for CALM and μ2, respectively. The fluorescence traces have a characteristic sigmoidal shape. (E) Montages of example clathrin-coated pit budding events. The blue arrow indicates t = 0 s, the moment of maximum decrease in CALM-GFP fluorescence that corresponds to vesicle scission. (F) Average CALM-GFP and μ2-mCherry at budding clathrin-coated pits. The average fluorescence traces were normalized to the average of the first three and last three fluorescence values for CALM and μ2, respectively. The μ2-mCherry fluorescence starts to decrease ∼15 s before the CALM-GFP signal (black arrow). (G) Heliquest server (http://heliquest.ipmc.cnrs.fr) helical wheel shows the orientation of the key hydrophobic residues (in yellow) and predicts the N terminus of CALM is an amphipathic helix with curvature-sensing properties. The hydrophobic moment points up toward the membrane and polar but mainly uncharged side chains are disposed laterally along where the surface the membrane would be. The negative charge points down away from the membrane. (H) Liposome-based SPR sensorgrams showing binding of CALM ANTH-WT, CALM ANTH(ΔH0), CALM ANTH(H0mut), and CALM ANTH(PIP−) to PtdIns4,5P2-containing liposomes at a protein concentration of 6 μM. (I) KD values for the binding of CALM ANTH domains, determined by liposome-based SPR (mean and SD of four independent measurements). Representative sensorgrams used for the measurements are shown in Figure S2A. We then compared the effects of expressing endogenous levels of full-length, siRNA-resistant CALM-wild-type (WT), CALM(PIP−), a mutant which has no PtdIns4,5P2 binding due to the mutations Lys28Glu, Lys38Glu, and Lys40Glu (Ford et al., 2001Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.Science. 2001; 291: 1051-1055Crossref PubMed Scopus (602) Google Scholar) and is therefore cytosolic or CALM(VAMP−), which has no R-SNARE binding due to the mutation Met244Lys (Miller et al., 2011Miller S.E. Sahlender D.A. Graham S.C. Höning S. Robinson M.S. Peden A.A. Owen D.J. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.Cell. 2011; 147: 1118-1131Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) in conjunction with endogenous CALM depletion (Figures S1A and S1B). Immunofluorescence indicated no obvious reduction in the number of punctate endocytic CCPs/CCVs (Figure S1A), whereas EM analysis of the CCP/CCV profiles showed that the expression of CALM-WT or CALM(VAMP−) rescued the relative ratios of open versus closed and sizes of CCPs/CCVs, whereas re-expression of CALM(PIP−) did not (Figures 1A and S1D). These data therefore indicate that CALM is not a major factor in CCP initiation, but rather that its presence strongly affects aspects of early to middle stages of CCP/CCV formation, and consequently influences the final CCV size. Because both CALM-WT and CALM(VAMP−) rescue phenotypes similarly, we further suggest that CALM’s SNARE binding is not a checkpoint during CCP/CCV formation. To substantiate our findings, we assessed the relative dynamics of AP2 and CALM during CCP formation in living cells with TIRF microscopy. These data showed that AP2 levels begin to decrease 15–20 s prior to the sharp decrease in the CALM levels that corresponds to the point when CCV scission occurs (Figures 2C–2F; see Cocucci et al., 2012Cocucci E. Aguet F. Boulant S. Kirchhausen T. The first five seconds in the life of a clathrin-coated pit.Cell. 2012; 150: 495-507Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, Loerke et al., 2009Loerke D. Mettlen M. Yarar D. Jaqaman K. Jaqaman H. Danuser G. Schmid S.L. Cargo and dynamin regulate clathrin-coated pit maturation.PLoS Biol. 2009; 7: e57Crossref PubMed Scopus (156) Google Scholar, Taylor et al., 2011Taylor M.J. Perrais D. Merrifield C.J. A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.PLoS Biol. 2011; 9: e1000604Crossref PubMed Scopus (515) Google Scholar, Traub, 2011Traub L.M. Regarding the amazing choreography of clathrin coats.PLoS Biol. 2011; 9: e1001037Crossref PubMed Scopus (37) Google Scholar). Within these 20 s prior scission, net membrane curvature must still increase to produce a necked and bulbous structure; hence, it is the level of CALM and not that of AP2 that correlates best with the increasing amount of positive curvature during CCP/CCV formation. CALM’s binding partners FCHO, NECAP, and clathrin (Ritter et al., 2013Ritter B. Murphy S. Dokainish H. Girard M. Gudheti M.V. Kozlov G. Halin M. Philie J. Jorgensen E.M. Gehring K. McPherson P.S. NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.PLoS Biol. 2013; 11: e1001670Crossref PubMed Scopus (42) Google Scholar, Tebar et al., 1999Tebar F. Bohlander S.K. Sorkin A. Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic.Mol. Biol. Cell. 1999; 10: 2687-2702Crossref PubMed Scopus (240) Google Scholar, Umasankar et al., 2012Umasankar P.K. Sanker S. Thieman J.R. Chakraborty S. Wendland B. Tsang M. Traub L.M. Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning.Nat. Cell Biol. 2012; 14: 488-501Crossref PubMed Scopus (63) Google Scholar) also bind AP2 and so should be present in CCPs even when CALM is absent. This suggests that CALM’s effects on CCP/CCV formation are not indirect through the action of these binding partners, but are consistent with a direct effect of CALM itself. The impact of CALM on the size and shape of CCPs/CCVs led us to re-examine the structure of CALM to determine how it could directly affect membrane curvature. Inspection of the N-terminal residues 1–18 of CALM with the Heliquest server (http://heliquest.ipmc.cnrs.fr) suggested that this region could, in fact, assume an amphipathic helix (AH) (Figure 2G). Membrane-interacting AHs adopt their helical conformations when they attach to phospholipid membranes (Bhatia et al., 2009Bhatia V.K. Madsen K.L. Bolinger P.Y. Kunding A. Hedegård P. Gether U. Stamou D. Amphipathic motifs in BAR domains are essential for membrane curvature sensing.EMBO J. 2009; 28: 3303-3314Crossref PubMed Scopus (201) Google Scholar, Drin et al., 2007Drin G. Casella J.F. Gautier R. Boehmer T. Schwartz T.U. Antonny B. A general amphipathic α-helical motif for sensing membrane curvature.Nat. Struct. Mol. Biol. 2007; 14: 138-146Crossref PubMed Scopus (439) Google Scholar, Gallop et al., 2006Gallop J.L. Jao C.C. Kent H.M. Butler P.J. Evans P.R. Langen R. McMahon H.T. Mechanism of endophilin N-BAR domain-mediated membrane curvature.EMBO J. 2006; 25: 2898-2910Crossref PubMed Scopus (418) Google Scholar); and indeed, liposome-based surface plasmon resonance (SPR) measurements showed that this potential N-terminal AH of CALM (we now term residues 1–18 as AH0, to be consistent with previous literature on other clathrin adaptors) contributed to the membrane binding of CALM. CALM ANTH bound to PtdIns4,5P2 liposomes at physiological salt concentration (170 mM) with a KD of 1.5 μM, whereas deletion of AH0 (CALM ANTH[ΔH0]), or the mutation of the key hydrophobic AH0 residues Leu6, Ile10, and Val17 to serines (CALM ANTH[H0mut]) both led to a decrease in affinity by ∼8-fold (Figures 2H, 2I, and S2A). Furthermore, in the presence of PtdIns4,5P2-containing liposomes, WT CALM ANTH showed a small but reproducible increase in α-helical content by circular dichroism (CD) (Figure S2B). Comparison of our CALM ANTH:VAMP8 complex structure (Miller et al., 2011Miller S.E. Sahlender D.A. Graham S.C. Höning S. Robinson M.S. Peden A.A. Owen D.J. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.Cell. 2011; 147: 1118-1131Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) with previous structures of the human and Drosophila CALM ANTH domains (Ford et al., 2001Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.Science. 2001; 291: 1051-1055Crossref PubMed Scopus (602) Google Scholar, Mao et al., 2001Mao Y. Chen J. Maynard J.A. Zhang B. Quiocho F.A. A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis.Cell. 2001; 104: 433-440Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Miller et al., 2011Miller S.E. Sahlender D.A. Graham S.C. Höning S. Robinson M.S. Peden A.A. Owen D.J. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.Cell. 2011; 147: 1118-1131Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) showed that residues 5–14 can indeed form a short N-terminal helix that is connected to helix1 by a linker with the central axes of the helices diverging by ∼40° at residues 14–17 (Figures 3A, 3E, S3A, and S3B). The structure of a C-terminally truncated form of the ANTH domain of CALM (CALM ANTH(1–264)) (Table S1; Figures 3C, S3A, and S3C) although lacking the SNARE binding site, again showed residues 5–18 form a helix but this time extending helix1 by an additional four turns (Figures 3C, 3E, S3A, and S3C). The increased stabilizing energy that would arise from the continued α-helical H-bonding pattern seen in the structure of CALM ANTH(1–264) suggests that this extension of helix1 is the most likely conformation for residues 5–18 when the helix formation is induced by either membrane insertion or by fortuitous crystal packing. The failure to detect the existence and effects of this helix, in previous in vitro studies, likely results from this region’s high proteolytic sensitivity. In other peripheral membrane proteins such as ArfGAP1, membrane-interacting amphipathic helices show membrane curvature sensing (MCS) and are referred to as ALPS (amphipathic lipid packing sensing) helices (Antonny, 2011Antonny B. Mechanisms of membrane curvature sensing.Annu. Rev. Biochem. 2011; 80: 101-123Crossref PubMed Scopus (309) Google Scholar, Bigay et al., 2003Bigay J. Gounon P. Robineau S. Antonny B. Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature.Nature. 2003; 426: 563-566Crossref PubMed Scopus (262) Google Scholar, Drin et al., 2007Drin G. Casella J.F. Gautier R. Boehmer T. Schwartz T.U. Antonny B. A general amphipathic α-helical motif for sensing membrane curvature.Nat. Struct. Mol. Biol. 2007; 14: 138-146Crossref PubMed Scopus (439) Google Scholar, Hatzakis et al., 2009Hatzakis N.S. Bhatia V.K. Larsen J. Madsen K.L. Bolinger P.Y. Kunding A.H. Castillo J. Gether U. Hedegård P. Stamou D. How curved membranes recruit amphipathic helices and protein anchoring motifs.Nat. Chem. Biol. 2009; 5: 835-841Crossref PubMed Scopus (286) Google Scholar). MCS arises from a combination of physicochemical effects including the insertion of the hydrophobic residue side chains of the helix into curvature-induced defects in membrane lipid packing and the interactions between the hydrophilic amino acid side chains of the helix and membrane phospholipid headgroups (Jensen et al., 2011Jensen M.B. Bhatia V.K. Jao C.C. Rasmussen J.E. Pedersen S.L. Jensen K.J. Langen R. Stamou D. Membrane curvature sensing by amphipathic helices: a single liposome study using α-synuclein and annexin B12.J. Biol. Chem. 2011; 286: 42603-42614Crossref PubMed Scopus (94) Google Scholar). When used in a sensitive microscope-based MCS assay on individual fluorescent liposomes (Lohr et al., 2009Lohr C. Kunding A.H. Bhatia V.K. Stamou D. Constructing size distributions of liposomes from single-object fluorescence measurements.Methods Enzymol. 2009; 465: 143-160Crossref PubMed Scopus (28) Google Scholar), CALM ANTH showed a preference for binding to small liposomes <100 nm diameter (around the size of closing CCPs), demonstrated by the enlarged population of smaller liposomes with higher protein densities (Figures 4A and S4A–S4G). CALM ANTH(ΔH0) showed a significantly reduced ability to sense curvature (Figures 4A and S4A–S4G), with a tendency to populate markedly lower protein densities. Taken together, our in vitro data indicate that CALM indeed possesses MCS capability, which is largely mediated by its AH0. The deformation/tubulation of liposomes is often used as an indicator of membrane sculpting activity in vitro (Itoh et al., 2005Itoh T. Erdmann K.S. Roux A. Habermann B. Werner H. De Camilli P. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.Dev. Cell. 2005; 9: 791-804Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, Peter et al., 2004Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.Science. 2004; 303: 495-499Crossref PubMed Scopus (1348) Google Scholar, Stachowiak et al., 2012Stachowiak J.C. Schmid E.M. Ryan C.J. Ann H.S. Sasaki D.Y. Sherman M.B. Geissler P.L. Fletcher D.A. Hayden C.C. Membrane bending by protein-protein crowding.Nat. Ce" @default.
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• W2097138052 title "CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature" @default.
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