Abstract
Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.
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Data availability
Coordinates and structure factors were deposited in the PDB with accession codes: 6QGS for WT/hAPT1; 6QGQ for C2S/hAPT1; 6QGO for S119A/hAPT1 and 6QGN for the 2Br-PLM-C2S/hAPT1 complex. Source data are provided with this paper.
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Acknowledgements
We thank D. Demurtas and G. Knott from the BioEM EPFL Core Facility for the electron microscopy analysis, S. Vossio and D. Moreau from the ACCESS Geneva screening platform; L. Menin from EPFL ISIC proteomic facility and the beamline scientists of X06DA at SLS (Villigen Switzerland). We thank all the members of the F.G.v.d.G. laboratory for their discussions and suggestions. This work was supported by the Swiss National Science Foundation, the Swiss National Centre of Competence in Research Chemical Biology and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreement no. 340260-PalmERa.
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L.A., M.A., P.A.S., M.D.P. and F.G.V.D.G. designed the concept. L.A., M.A., S.H., M.J.M., M.U.A., P.A.S., G.F. and F.P. conducted the investigation. M.D.P. and F.G.V.D.G. acquired the funding. M.A., L.A., S.H., M.J.M., M.D.P. and F.G.V.D.G. wrote the original draft. L.A., M.A., S.H., M.J.M., M.U.A., P.A.S., G.F., F.P., M.D.P. and F.G.V.D.G. reviewed and edited the manuscript. S.H. and L.A. were responsible for obtaining resources.
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Extended data
Extended Data Fig. 1 The APT crystallographic dimers.
a, Comparative superimposition of S119A APT1/6QGO (light-blue), WT APT1/6QGS (green) and WT APT1/1FJ2 (dark-pink). The average backbone RMSD value is 24 Å. b, Principal component analysis based on atomistic MD simulations on the WT APT1; c, Comparative superimposition of WT APT2/5SYN (orange) and WT APT2/6BJE (light yellow). The average backbone RMSD value is 14 Å; d, Superimposition of WT APT1/6QGS (green) and WT APT2/5SYN (Orange). The average backbone RMSD value is 20 Å. In surface blue representation are the catalytic residues: S119, D74 and H208. e, Association of WT APT1 and β-tongue mutant with liposomes. WT and mutant APT1 proteins were incubated with liposomes and loaded on the bottom of a sucrose gradient. The different interfaces from the top (1) to bottom were collected, loaded on an SDS-PAGE gel, and revealed with Coomassie blue. f, The thioesterase activity of WT APT2, the PosPatch or the ß tongue mutants was monitored as a function of time after the addition of substrate and detergent. APT2-specific inhibitor ML349 was included as a positive control. Technical replicates were averaged within each experiment. Average of the activity between multiple independent experiments at time 60 min are shown in Fig. 2.
Extended Data Fig. 2 WT and palmitoylation deficient APT2.
HeLa cells were transfected with different myc-tagged APT2 constructs for 24 h. a, Cells were then metabolically labeled for 3 h at 37 °C with 3H-palmitic acid. Proteins were extracted, immunoprecipitated with anti-myc antibodies, subjected to SDS-PAGE gel, analyzed by autoradiography (3H-palm), and quantified using the Typhoon Imager or by immunoblotting with anti-myc antibodies. The calculated value of 3H-palmitic acid incorporation into WT APT2 was set to 100%, and the values for the mutants were expressed relative to this (results are mean±SD, n = 5 independent experiments). b, Cells were pulsed with 35S Cys/Met for 20 min and were chased for the indicated time before immunoprecipitation and SDS-PAGE. Degradation kinetics were analyzed by autoradiography, and were quantified using the Typhoon Imager. 35S-Met/Cys incorporation was quantified for each time point. 35S-Met/Cys incorporation was set to 100% for t = 0 after the 20 min pulse, and the different chase times were expressed relative to this (results are mean±SD, n = 5 independent experiments). c, Cells were incubated with 1 µM of Palmostatin B for different times at 37 °C, lysed, subjected to SDS-PAGE, and analyzed by immunoblotting with anti-myc antibodies. d, Thermal denaturation profiles of WT APT2 protein alone or associated with liposomes (interface 1 of sucrose gradient) as monitored by circular dichroism at 222 nm. The temperature of the samples was increased from 4 °C to 94 °C by 2 °C intervals. The normalized ellipticity at 222 nm is plotted against temperature, results are mean±SD, n = 3 technical replicates. e, The levels of APT1 and 2 mRNA were determined by qPCR upon silencing of APT1 or 2, or various ZDHHC genes.
Extended Data Fig. 3 Effect of S-acylation on APT2 localization and stability.
a-e : HeLa cells were transfected with plasmids encoding WT or C2S APT2 for 24 h. a, Confocal microscopy images of cells expressing WT or C2S APT2-myc immunolabeled for APT2 and Giantin. Nuclei were stained with Hoechst. Scale bar: 10 µm. b, PNSs were prepared and ultra-centrifuged to separate the membrane (Pellet) and cytosolic (Sup.) fractions. Equal volumes were analyzed by SDS-PAGE. APT2 WT or C2S levels in each fraction were normalized to that in the PNS (results are mean±SD, n = 3 independent experiments). c, Cells expressing APT2-myc constructs were treated or not for 4 h with Palmostatin B, pulsed with 35S Cys/Met for 20 min and then chased for the indicated time before immunoprecipitation and SDS-PAGE. 35S-Met/Cys levels were determined for each time point by autoradiography and quantified using the Typhoon Imager. 35S-Met/Cys levels were normalized to that at t = 0 after the 20-min pulse (results are mean±SD, n = 3 independent experiments). d, PNS were prepared from cells expressing WT or C2S APT2-myc and ultra-centrifuged to separate membrane (Pellet) and cytosolic (Sup.) fractions. The amount of palmitoylated protein was determined using Acyl-RAC. For each fraction, palmitoylated proteins were detected after hydroxylamine treatment (+HA). Equal volumes were by analyzed SDS-PAGE and immunoblotting with anti-GFP antibodies. e, Cells were treated for 4 h with MG132 and were then metabolically labeled for 3 h at 37 °C with 3H-palmitic acid. The proteins were extracted, immunoprecipitated with anti-myc antibodies, separated via SDS-PAGE, and analyzed by autoradiography (3H-palm), which was quantified using the Typhoon Imager or by immunoblotting with anti-myc antibodies. The calculated value of 3H-palmitic acid incorporation into WT APT2 was set to 100%, and mutants were expressed relative to this (results are mean±SD, n = 3 independent experiments).
Extended Data Fig. 4 Identification of the APT2 acyltransferases.
a, HeLa cells were silenced for 3 days with individual or mixed pools of ZDHHC RNAi transfected with plasmids encoding citrine-tagged WT APT2. Cells were immunolabeled for citrine-APT2 (green). Bar 10 µM. b, HeLa cells, silenced for 3 days using a control siRNA or an siRNA against ZDHHC3 were immunolabeled against endogenous ZDHHC3 or the Golgi marker GM130. Bar 10 µM. c, HeLa cells expressing myc-ZDHHC7 were immunostained against myc and the ER marker Bap31. Bar: 10 µM. d, The kinetics of recovery after photobleaching were determined for cytosolically expressed mCitrine (~27 kDa) and mCherry-mCitrine (~55 kDa). T1/2 were computed from FRAP curves using non-linear regression assuming one-phase association. Results are mean±SEM, n = 8 independent experiments. The recovery time was not affected by the molecular weight difference.
Extended Data Fig. 5 Palmitate binding in the APT hydrophobic pocket.
a, Ribbon diagrams of APT1 WT and mutants showing the palmitate moiety in blue into the catalytic pocket with the Fo-Fc map in grey mesh. b, APT1 WT showing the 2-bromopalmitate (2-BP) moiety in yellow into the catalytic pocket with the Fo-Fc map in grey mesh. The Br Fo-Fc is displayed with a red mesh. b, Determination of the effect of 2-BP on the thioesterase activity of WT APT1 and WT APT2 at 60 min after the addition of substrate and detergent. APT1-specific inhibitor ML348 or APT2-specific inhibitor ML349 at 10 µM were included as positive and negative controls, results are mean±SD, n = 3 technical replicates. c,d, HeLa cells were transfected with plasmids encoding myc-tagged WT ZDHHC6 constructs for 24 h. Cells were metabolically labeled for 2 h at 37 °C with 3H-palmitic acid and were chased for different times in new complete medium in the presence or not of 2-BP only during the chase. Proteins were extracted, immunoprecipitated with anti-myc antibodies, subjected to SDS-PAGE, analyzed by autoradiography (3H-palm), and quantified using the Typhoon Imager or by immunoblotting with anti-myc antibodies. 3H-palmitic acid incorporation was set to 100% for cells after the pulse, and values obtained after different chase times were expressed relative to this (results are mean±SD, n = 3 independent experiments). e, 2-bromopalmitate was bound in all APT1 subunits in the asymmetric unit. Ribbon diagram of the 2-BP/APT1 asymmetric unit. The 2-BP molecules are shown in sticks. f, Ribbon diagram of the side (left) and front (right) view of the APT1 enzyme. The residues forming the binding pocket are shown in sticks representation: in red the residues forming the entrance of the pocket, in blue the residues composing the top of the pocket and in light-blue the residues defining the end of the pocket. The rest of the channel is formed by the residues in pink. The entrance of the channel is solvent-exposed and indicated with an orange arrow. The palmitic acid is shown as blue sticks. g, Ribbon diagram of the front view of the apo WT APT1 enzyme (light-green) and palmitate-bounded WT APT1 enzyme (dark-green). In sticks representation, the residues involved in the regulation of the lipid access: Leu184, Phe181, and Leu78.
Extended Data Fig. 6 The APT2 hydrophobic pocket is essential for activity.
a, Level of expression of APT2 pocket mutants. Total cell extract (TCE) from cells expressing WT or mutant APT2 for 24 h were subjected to SDS-PAGE, and analyzed by immunoblotting with anti-myc antibodies. Anti-actin antibodies were used as a loading control. b, HeLa cells were transfected 24h with plasmids encoding the indicated APT2 constructs. Cells were then metabolically labeled for 2 h at 37 °C with 3H-palmitic acid. APT2 was immunoprecipitated with anti-myc antibodies, subjected to SDS-PAGE, immunoblotted with anti-myc antibodies, and analyzed by autoradiography (3H-palm). Quantification of the 3H-palmitic acid incorporation into different APT2 mutants. The calculated value of 3H-palmitic acid incorporation into WT APT2 was set to 100%, and the mutants were expressed relative to this (results are mean±SD, n = 3 independent experiments). c, ZDHHC6 palmitoylation upon overexpression of APT2 pocket mutants. HeLa cells were silenced for 3 days with APT2 RNAi and were transfected with plasmids encoding myc-tagged WT ZDHHC6 and the indicated APT2 constructs for 24 h. The cells were then metabolically labeled for 2 h at 37 °C with 3H-palmitic acid and were chased for 3 h. Proteins were extracted, immunoprecipitated with anti-myc antibodies, separated via SDS-PAGE, immunoprecipitated with anti-myc antibodies, and analyzed by autoradiography (3H-palm). The total extracts (40 µg) were immunoblotted with anti-myc antibodies to determine the expression level of WT and mutant APT2. The calculated value of 3H-palmitic acid incorporation into ZDHHC6 with WT APT2 was set to 100%, and the mutants were expressed relative to this (results are mean±SD, n = 3 independent experiments). d, Representative snapshot of the membrane-bound APT2 state. In orange, the APT2 protein. The catalytic pocket is shown as blue surface, and the membrane bilayer in grey.
Supplementary information
Supplementary Table
Crystallographic data table.
Source data
Source Data Fig. 1
SEC–MALS ultraviolet and molecular weight data.
Source Data Fig. 2
CD wavelength scan and temperature melt data; activity assay WT-PosPatch-bTongue mutants.
Source Data Fig. 2
Unprocessed SDS–PAGE gels, liposome binding assays. Uncropped electron microscopy images.
Source Data Fig. 3
Pulse-chase data.
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 4
Ratio data, 3H-incorporation data, FRAP data.
Source Data Fig. 4
Unprocessed autoradiographs and unprocessed western blots.
Source Data Fig. 5
3H-incorporation and pulse-chase data.
Source Data Fig. 5
Unprocessed autoradiographs and unprocessed western blots.
Source Data Fig. 6
Activity assay WT-binding pocket mutants.
Source Data Extended Data Fig. 1
Activity assay WT-bTongue mutants.
Source Data Extended Data Fig. 1
Unprocessed SDS–PAGE gels and liposome binding assay.
Source Data Extended Data Fig. 2
3H- and 35S -incorporation pulse-chase data, CD temperature melt data, RNA level data.
Source Data Extended Data Fig. 2
Unprocessed autoradiographs and unprocessed western blots.
Source Data Extended Data Fig. 3
Quantification PNS-P-S, 3H- and 35S: incorporation pulse-chase data.
Source Data Extended Data Fig. 3
Unprocessed autoradiographs and unprocessed western blots.
Source Data Extended Data Fig. 4
FRAP data.
Source Data Extended Data Fig. 5
3H-incorporation pulse-chase data, activity assay 2BP.
Source Data Extended Data Fig. 5
Unprocessed autoradiographs and unprocessed western blots.
Source Data Extended Data Fig. 6
3H-incorporation data.
Source Data Extended Data Fig. 6
Unprocessed autoradiographs and unprocessed western blots.
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Abrami, L., Audagnotto, M., Ho, S. et al. Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains. Nat Chem Biol 17, 438–447 (2021). https://doi.org/10.1038/s41589-021-00753-2
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DOI: https://doi.org/10.1038/s41589-021-00753-2
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