bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Eliot Morrison1, Tatjana Wegner1, Andres Ernesto Zucchetti2, Miguel Álvaro-Benito1, Ashley Zheng1, 3 4 5 2 1* Stefanie Kliche , Eberhard Krause , Britta Brügger , Claire Hivroz & Christian Freund

1Freie Universität Berlin, Institute for Chemistry & Biochemistry, Laboratory of Biochemistry, Thielallee 63, 14195 Berlin

2Institut Curie, PSL Research University, INSERM U932, Integrative analysis of T cell activation team, 26 rue d'Ulm, 75248, Paris Cedex 05, France

3Otto-von-Guericke-University, Institute of Molecular and Clinical Immunology, Health Campus Immunology, Infectiology and Inflammation, Leipziger Strasse 44, 39120 Magdeburg, Germany

4Leibniz Institute for Molecular Pharmacology, Mass Spectrometry Unit, Robert-Rössle-Str 10, 13125 Berlin

5Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120, Heidelberg, Germany.

*Corresponding address: [email protected]

Abstract Palmitoylation is the reversible addition of palmitate to via a thioester linkage. Following stimulation of the T-cell receptor we find a number of are newly palmitoylated, including those involved in vesicle- mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On- Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.

Keywords: Palmitoylation, S-acylation, TCR Signaling, T-Cell Activation, Palmitome, In vitro palmitoylation, VAMP7, Ti-VAMP, LAT, DHHC PATs, DHHC18, DHHC20

1 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The initial signaling events following the recognition of a peptide-MHC complex on an antigen-presenting cell (APC) by the T-cell receptor (TCR) involve a number of important molecules that must localize to the plasma membrane (PM) at the immunological (IS). Palmitoylation, the reversible linking of 16-carbon palmitic acid to via a labile thioester bond, is one strategy used by several important TCR-signaling proteins to drive this localization to and from the membrane (e.g. the soluble Src-family kinases and Fyn) or to and from liquid-ordered microdomains (“lipid rafts”) within the membrane (e.g. the transmembrane scaffold LAT)1. Unlike other lipid modifications like or prenylation, the reversible chemistry of the thioester bond allows palmitoylation to be both added to and removed from specific cysteines; indeed, have evolved to both add (e.g. the DHHC family of protein acyltransferases [PATs]) and remove (e.g. acyl protein thioesterase 1 [APT1] and ABHD17) palmitate from these sites in eukaryotes2,3. This has given rise to palmitoylation cycles, where proteins are palmitoylated in the ER/Golgi, cycled to the PM via vesicle trafficking and localized to the site of activity, then depalmitoylated and recycled back to the ER/Golgi4. Such cycles have become well-established in neuronal proteins like PSD-95, as well as the G-protein α 4,5 subunits Gαi and Gαq and small GTPases H-Ras and N-Ras , and pulse-chase experiments have shown that palmitoylation turnover can be accelerated following signaling stimuli, such as glutamate receptor activation with PSD-95, GTP binding of H-Ras, and GPCR signaling for Gα proteins6–8. In the context of T-cells, Lck has been shown to undergo relatively rapid palmitate turnover under resting conditions, which is accelerated following Fas receptor stimulation, and this palmitoylation is specifically catalyzed by DHHC21 at the PM9. A decrease in the palmitoylation of LAT has been observed in anergic T cells, which affects its ability to localize to lipid rafts and induce an effective stimulatory response1,10. Docking of vesicular LAT to the IS and downstream signaling also critically depend on palmitoylated SNARE proteins like SNAP-23 and VAMP711,12. While these targeted approaches suggest the possibility of regulated palmitoylation cycles, a global approach tracking changes of the complete palmitome of T-cells following a stimulatory event has not been reported. Equally enigmatic are the enzymes responsible for catalyzing the palmitoylation reaction, the DHHC family of PATs, first identified in yeast more than 15 years ago13,14. While 23 PATs are expressed in , due to the challenges inherent in expressing and purifying these multi-pass membrane proteins, only a handful of mammalian PATs have been investigated in vitro: the palmitoylation of Gαi by DHHC2 and DHHC3, the palmitoylation of PSD-95 by DHHC15, and the autopalmitoylation of DHHC9 and DHHC2015–19. These studies often rely on the use of [3H]-palmitic acid to quantify the palmitoylated substrate, requiring long exposure times and the hazards of radioactive materials, or else are limited to the kinetics of the autopalmitoylation reaction using -linked assays or palmitoylation targets of peptide length. We here developed the On-Plate Palmitoylation Assay (OPPA) method as a rapid, click chemistry-based approach to quantifying an intact palmitoylated substrate over time. The understanding of palmitoylated SNARE proteins has generally been restricted to those lacking transmembrane domains (TMDs), such as SNAP-25 and the vesicular SNARE Ykt6, where the addition of hydrophobic palmitate assists in membrane recruitment20. However, the vesicular -family SNARE protein VAMP7, despite featuring a single TMD at its C-terminus, has been reported to be palmitoylated in a large number of different mammalian global palmitome studies21–30. The observation that many of the proteins found to be palmitoylated are in fact integral membrane proteins (up to ~50-60% of proteins enriched by ABE) suggests the 2 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

mechanism of regulated palmitoylation is subtler than the simpler model of membrane recruitment31. Possible reasons why a transmembrane protein might be palmitoylated include the mediation of protein-protein interactions within the membrane, the induction of conformational changes (e.g. “tilting” within the membrane or the recruitment of soluble tails to the membrane), the driving of localization to liquid-ordered microdomains within the membrane, or as an interplay with other post-translation modifications like 32. The palmitoylation of VAMP7 might represent a new mode of transmembrane SNARE protein palmitoylation beyond the relatively straightforward membrane recruitment seen in the soluble SNARE proteins, allowing an additional layer of subtlety to its regulation.

Results and Discussion The composition of the T-cell palmitome changes upon TCR stimulation. Using the acyl-biotin exchange (ABE) method with SILAC-labeled Jurkat T cell lysates allowed us to quantify the T-cell palmitome via quantitative LC-MS/MS (Fig. 1A)31. We previously reported the ABE-enriched Jurkat palmitome under resting conditions31; here we re-analyze these data with a volcano plot-based approach, allowing us to identify 115 high-confidence palmitoylation candidates, including well-established palmitoylated proteins like Lck and LAT (Fig. 1B). This allows for the direct comparison to the Jurkat palmitome 10 minutes following anti-CD3/anti-CD28 costimulation (Fig. 1C and D, Supp. Fig. 1). After stimulation, 169 proteins are found in the high-confidence palmitoylated pool. Three criteria were used for validation (Fig. 1E): number of previous reports in 13 palmitome studies, number of predicted palmitoylation sites (CSSPalm 4.0), and number of predicted transmembrane domains (TMDs) (TMHMM 2.0); we see an overrepresentation of all three criteria in our enriched pools versus background unenriched proteins, confirming the enrichment27,28,39–43,30,31,33–38. Directly comparing these two pools of proteins reveals 94 constitutively palmitoylated proteins, 18 stimulation-dependent depalmitoylated proteins, and 70 stimulation-dependent modified proteins (Supp. Table 1). Lck and LAT are constitutively palmitoylated, while among the stimulation-dependent palmitoylated proteins are the v-SNARE protein VAMP7 and the PAT DHHC18. VAMP7 belongs to the synaptobrevin family of v-SNAREs, and has been shown to be crucial for docking vesicular LAT to the IS and downstream signaling events in CD4+ T cells11. DHHC18 is a Golgi-localized PAT, and although it has been described to palmitoylate H-Ras44,45, little is known about its enzymatic properties or substrate specificity19. As autopalmitoylation is the initial step in the PAT enzymatic mechanism46, finding this PAT palmitoylated after TCR stimulation presumably represents an activated state of this enzyme, and may hint at an upregulation of specific PAT activity following TCR stimulation. Of the stimulation-dependent palmitoylation candidates, approximately half are transmembrane proteins. Grouping the protein interaction networks using STRING analysis reveals a large cluster of Gα, Ras, and Ras- related proteins (Fig. 2A). Intriguingly, within this cluster are GNAI3, GNAO1, and GNAI1/2, all of which feature N-terminal myristoylation motifs, while the Gα proteins GNAQ and GNA13, both of which lack a myristoylation motif, are found to be constitutively palmitoylated. This could suggest an interplay between co-translational myristoylation and post-translational, stimulation-dependent palmitoylation, in which myristoylated Gα proteins are preferentially palmitoylated following TCR stimulation. Indeed, the stimulation-dependent palmitoylation targets feature a slight enrichment of proteins containing myristoylation motifs (8.8%) versus constitutively palmitoylated proteins (6.4%), perhaps hinting at a larger trend. Functionally, the stimulation-dependent 3 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

palmitoylation candidates are involved in a variety of cellular processes (Fig. 2B), notably Ras signal transduction, TCR signaling, and vesicle-mediated transport.

The OPPA method allows for rapid determination of steady-state palmitoylation rates. To investigate these stimulation-dependent palmitoylation candidates in a controlled setting, we designed constructs of DHHC18, DHHC20, VAMP7, and LAT for expression in Sf9 insect cells (Fig 2A, Supp. Figs. 4,5). Although VAMP7 has been reported as palmitoylated in eight other human palmitome studies23,24,27,28,30,38,39,43,47, the site of its palmitoylation has not been described. Of the six cysteines in its primary sequence, we found three NEM-modified during our ABE enrichment (data not shown), suggesting they are not palmitoylated, while two others are located in the vesicular lumen, not accessible to a DHHC PAT’s cytosolic active site; the remaining cysteine, Cys183, which is located in an optimal juxtamembrane position, was therefore chosen for mutational studies as the most likely site of palmitoylation33. Likewise, in DHHC18, we sought to investigate the role the N- and C-terminal tails of DHHC18 might play in the enzyme’s stability or activity by designing ΔN and ΔC domain constructs. Moreover, we designed a construct based on the truncated isoform 2 of DHHC18, which we previously hypothesized was preferentially palmitoylated in T cells31. Intriguingly, this isoform lacks the two N-terminal TMDs of the full-length protein, yet features a cysteine (Cys138) only three residues from its new N-terminus, which might represent a palmitoylation anchor to the membrane. In order to improve upon the shortcomings of current in vitro palmitoylation assays, we developed the On-

Plate Palmitoylation Assay (OPPA) method (Fig 2B). Briefly, purified substrates (e.g. VAMP7) bearing both His6- and FLAG-tags are bound to Ni-NTA plates, to which purified DHHC PATs (lacking His6-tags) and clickable palmitic acid alkyne (PAA)-CoA are added. The palmitoylation reaction is quenched at specific time points by N- ethylmaleimide (NEM), which both blocks the site of palmitoylation and the DHHC active-site cysteine. After washing, biotin-azide is clicked onto the palmitoylated substrate, which is subsequently eluted from the Ni-NTA plate and bound to anti-FLAG-coated plates. This allows the detection of the biotinylated substrate via fluorescence of Eu3+-streptavidin and the determination of the steady-state rate of palmitoylation.

Examining DHHC18 and DHHC20 activity by OPPA Using the VAMP7 and LAT constructs as palmitoylation targets and the DHHC18 and DHHC20 constructs as palmitoylating enzymes allowed us to validate the OPPA method and investigate these reactions in vitro (Fig 2C). While autopalmitoylation of VAMP7 is minimal at 20 µM PAA-CoA (Supp. Figs 6-8), the rate of palmitoylation is greatly enhanced by full-length DHHC18. The active-site mutant DHHC18-C222A prevents this palmitoylation. DHHC20 was barely capable of palmitoylating VAMP7, while its active-site mutant C156A is also inactive. Truncating either the N- or C-terminal tails of DHHC18 results in a significant decrease in the palmitoylation rate of VAMP7. Whether this speaks for a role in these cytosolic tails in substrate recognition or engagement, in its enzymatic mechanism, or in simply stabilizing the enzyme will require further investigation. The short isoform DHHC18-2, lacking the N-terminal TMDs, is capable of palmitoylating VAMP7, albeit at a slightly decreased rate. Intriguingly, mutating the proposed palmitoylation anchor cysteine C138 to alanine 4 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

prevents DHHC18-2’s palmitoylation of VAMP7, while this mutation in full-length DHHC18-1 has no effect; one potential model to explain this is shown in Figure 2B. Finally, LAT was used as a second palmitoylation target. Full-length DHHC18 was capable of palmitoylating LAT, while DHHC20 failed to do so. This may speak to a specificity for LAT palmitoylation by DHHC18 and not DHHC20. As DHHC20 is located at the PM and turnover of LAT palmitoylation is not expected to be rapid, the specificity of Golgi-localized DHHC18 could perhaps reflect a biological reality; this should be confirmed in a cellular context, perhaps with PAT-specific knockouts and subsequent palmitome enrichment, as was recently performed for DHHC1345,48.

VAMP7 is palmitoylated at Cys183 in vitro and in vivo and is required for Golgi localization The proposed palmitoylation site in VAMP7, Cys183, was also mutated to alanine. This prevented its palmitoylation by both DHHC18 and DHHC20, suggesting that this is indeed the primary site of in vitro palmitoylation. To confirm this in vivo, CRISPR/Cas9 knockout Jurkat cell lines with lentivirally transduced stable re-expression of VAMP7-WT and -C183A constructs were engineered (Supp. Fig. 10). Once generated, ABE was used to enrich all palmitoylated proteins in these cell lines. VAMP7 was found to be palmitoylated in the Jurkat E6.1 and VAMP7-WT re-expression cell lines, but not in the VAMP7 knockout or VAMP7-C183A re-expression cell lines, confirming that Cys183 is the site of VAMP7 palmitoylation in Jurkat T cells (Supp. Fig. 11). The VAMP7-KO Jurkat cell lines stably expressing VAMP7-WT and VAMP7-C183A were analyzed by TIRFM to observe localization of these two constructs. The re-expressed VAMP7-WT, which is capable of being palmitoylated, is largely found in the Golgi of the VAMP7 knockout/re-expression Jurkat cells, in agreement with earlier observations (Fig 4A)49. VAMP7-C183A, however, does not co-localize with the Golgi marker giantin, instead localizing to the cytosol, presumably within vesicles (Fig 3). This supports the conclusion that palmitoylation of VAMP7 at Cys183 acts as a sorting signal for the Golgi50,51. Mutating Cys183 prevents this localization, allowing a larger population of VAMP7 to localize to vesicles 11,52. Since VAMP7 has been shown to control membrane trafficking from and to the Golgi53, mislocalization of the mutant form of VAMP7 may have some consequences on the trafficking of cargoes in T lymphocytes. It might also alter some cell functions such as polarized transport of proteins or cell migration. The fact that, as shown here, localization of this key v-SNARE protein is regulated upon TCR triggering reinforces the link that exists between TCR-induced signaling and intracellular trafficking54.

Materials and Methods Jurkat Cell Culture and Stimulation Jurkat T cells (clone E6-1) were grown in RPMI 1640 SILAC medium with 10% FCS at 37 ˚C and 5%

CO2 (SILAC Quantification Kit, Pierce). Cells were stimulated using anti-CD3 IgM (C305) and anti-CD28 IgM hybridoma supernatants in RPMI without FCS at 37 ˚C; stimulation was quenched after 10 minutes by the addition of ice-cold PBS, centrifugation (300xg, 5 min, 4 ˚C), and subsequent shock-freezing of the cell pellets.

5 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Generation of VAMP7 Knockout in Jurkat Cells For the generation of sgRNAs the Atum online tool was used (https://www.dna20.com/ eCommerce/cas9/input). The sgRNA oligos were then cloned into the pSpCas9(BB)-2A-GFP (PX458) vector (a kind gift from Feng Zhang [Addgene plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138]). Jurkat T cells (clone E6-1) were passaged to 0.125 Mio cells/ml the day before transfection. 5 Mio cells were resuspended in 100µl OptiMem with 10 µg pSpCas9(BB)-2A-GFP with the sgRNA CACCGCACACCA- AGCATGTTTGGCA and electroplated with a NEPA21 electroporator (Nepagene) with the following settings: 0.2 mm gap electroporation cuvettes, poring pulse: 175 V, length: 5 ms, interval: 50 ms, number: 2, D. rate: 10%, polarity: +. After 48 hours single clones were sorted by FACS for EGFP into 96-well plates. After the cells had recovered and grown the knockout was validated by Western blotting and sequencing.

Lentiviral Transduction of Jurkat Cells Once the VAMP7 knockout cell line was validated, the re-expression of VAMP7 constructs was performed. Wildtype VAMP7 and mutant VAMP7-C183A, both featuring FLAG tags, were cloned into the lentiviral vector LegoiG2 (a kind gift from Boris Fehse [Addgene plasmid # 27341; http://n2t.net/addgene:27341; RRID:Addgene_27341])55. HEK293 cells were transfected with the constructs FLAG-VAMP7-WT-LegoiG2 or FLAG-VAMP7-C183A-LegoiG2 plus the third-generation packaging plasmids pMDLg/pRRE with pRSV-Rev and phCMV-VSV-G using lipofectamine. Supernatants containing viruses were harvested after 48 hours and concentrated by centrifugation at 10,000xg and 4°C for 4 to 6 hours in glass tubes. VAMP7 knockout Jurkat cells were transduced with the viruses by spinoculation, as described previously56. Cells were resuspended in lentiviral supernatant supplemented with Polybrene (6µg/ml) and spun for 90 min at 37°C at a speed of 800xg. After 72 hours the cells were sorted by EGFP expression via FACS. One week later the cells were sorted again for EGFP expression and also for the expression of CD3 and CD28 surface markers.

Construct Design and Cloning Primary sequences of VAMP7, LAT, DHHC18 and DHHC20 were obtained from UniProt. Full-length constructs of VAMP7 and LAT were engineered bearing His6- and FLAG-tags, while DHHC18 and DHHC20 were designed with FLAG tags alone, and ordered from Geneart. Truncated constructs of DHHC18 were generated by amplifying FLAG-tagged inserts via PCR. Mutagenesis was performed using the QuikChange Site-Directed Mutagenesis system (Agilent). Constructs were then cloned into the pFastBac1 vector of the Bac-to-Bac system (Thermo Fisher).

Insect Cell Expression and Purification Sf9 insect cells were grown in Sf-900 II medium (Thermo Fisher) at 27 ˚C. Baculoviruses were generated by transfecting insect cells with constructs in the pFastBac1 vector according to the Bac-to-Bac protocol. P4 viral stocks were then used to infect larger cultures, and expressed proteins were harvested 3-5 days following infection

(~70-80% cell viability). Cell pellets were shock frozen and lysed in lysis buffer containing 50 mM NaH2PO4 (pH 7.2) (Roth), 300 mM NaCl (Roth), 0.2% Triton X-100 (Roth), 1 mM phenyl methanesulfonyl fluoride (PMSF) (Sigma), cOmplete protease inhibitor cocktail (Roche), and DNase (New England BioLabs) and RNase (Pancreac 6 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

AppliChem) on ice for 30 minutes. Lysates were cleared by centrifugation (16,000xg, 4 ˚C, 10 min.) and supernatants were loaded onto anti-FLAG M2 affinity gel (Sigma) at 4 ˚C. After washing, proteins were eluted using 0.1 M glycine at pH 3.5 and concentrated using Vivaspin spin filters (VWR) with appropriate MW cut-offs. After concentration, 0.08% n-dodecyl β-D-maltoside (DDM) (Sigma) and cOmplete protease inhibitor cocktail was added and constructs were dialyzed overnight at 4 ˚C into 50 mM NaH2PO4 (pH 6.5) and 10% glycerol (Roth). Protein concentration was determined by BCA assay (Pierce) and expression was confirmed by anti-FLAG Western blot. Purified PATs were extremely sensitive to environmental conditions, precipitating at concentrations greater than ~5-10 µM and losing activity after freezing or 3-5 days storage at 4 ˚C. For this reason, all purified PATs were used immediately for the OPPA assays following dialysis.

Western Blotting To confirm the stimulation via phosphotyrosine and phospho-ERK1/2, and to confirm expression/purification of constructs expressed in insect cells, lysates were run on SDS-PAGE gels and transferred to nitrocellulose membranes using standard methods. used for detection: 4G10 Platinum anti- phosphotyrosine (Millipore), anti-pERK (E-4) (Santa Cruz), anti-β-actin (AC-15) (Sigma Aldrich), anti-LAT (Upstate (Millipore)) and anti-FLAG-HRP (Sigma Aldrich). The self-produced VAMP7 was a kind gift of Dr. Andrew Peden (The University of Sheffield, UK).

Acyl Biotin-Exchange The ABE protocol was followed as described previously31,57. Briefly, stimulated Jurkat SILAC pellets were lysed in lysis buffer containing 50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem),

10mM KCl (Sigma-Aldrich), 500 µM EDTA (Roth), 100 µM Na3VO4 (Sigma-Aldrich), 20 mM NEM (Thermo), 1 mM PMSF, 1.7% Triton X-100, 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 µM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), and cOmplete protease inhibitor cocktail (PI-cocktail) on ice for 30 minutes. Lysates were cleared by centrifugation (16,100xg, 4 ˚C, 10 min.). Chloroform-methanol (CM) precipitations were used between each chemical labeling step to ensure complete remove of unreacted reagents. The cleared lysates were subjected to a CM precipitation, then resuspended in buffer containing a 50 mM Tris (pH 7.4), 5 mM EDTA, 1% SDS (Roth), 125 mM NaCl, 1 mM PMSF, 0.2% Triton X-100, 20 mM NEM, and PI-cocktail and incubated overnight at 4 ˚C. NEM was then removed by three sequential CM precipitation steps, and enriched samples (e.g. heavy lysates) were resuspended in buffer containing 50 mM Tris (pH 7.4), 5 mM EDTA, 125 mM NaCl, 1% SDS, 574 mM hydroxylamine (HA) (Sigma-Aldrich), 820 µM EZ-Link HPDP-biotin (Thermo), 0.2% Triton X-100, 1 mM PMSF and PI-cocktail; control samples (e.g. light lysates) were resuspended in a similar buffer, omitting HA. After incubation for one hour at room temperature, a single CM precipitation removed HA, and samples were biotinylated in a buffer containing 50 mM Tris (pH 7.4), 5 mM EDTA, 125 mM NaCl, 164 µM EZ-Link HPDP-biotin, 0.2% Triton X-100, 1 mM PMSF and PI-cocktail for one hour at room temperature. Samples were then washed with three sequential CM precipitations and resuspended in buffer containing 50 mM Tris (pH 7.4), 5 mM EDTA, 125 mM NaCl, 0.1% SDS, 0.2% Triton X-100, 1 mM PMSF and PI-cocktail. SILAC-labeled heavy and light samples were then mixed and allowed to bind to streptavidin-agarose beads (Novagen) for 90 min at room temperature. After four sequential washings, biotinylated peptides were eluted 7 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

by cleaving the HPDP-biotin-cysteine linkage in a buffer containing 1% beta-mercaptoethanol (2-ME) (Roth) at 37 ˚C for 15 min. Eluates were precipitated via trichloroacetic acid (TCA) precipitation and resuspended in a smaller volume of SDS-PAGE sample buffer containing 0.2% Triton X-100, boiled for 5 min. at 95 ˚C, then run on a 4-12% Bis-Tris gel (NuPAGE Novex). After Coomassie staining, gels were used for in-gel tryptic digestion and subsequent LC-MS/MS. For the stimulated Jurkat cells, four individual ABE enrichments were performed (n=4).

Quantitative LC-MS/MS and Proteomic Data Analysis Coomassie-stained gel lanes from the ABE enrichment were cut into 30 equal-sized bands and in-gel tryptic digest was performed as described previously31. Digested peptides were resuspended in 6 µl 0.1% (v/v) TFA and 5% (v/v) acetonitrile per band. Analysis of the peptides was performed via reversed-phase capillary liquid chromatography (Ultimate 3000 nanoLC system (Thermo Scientific)) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific). LC separations were performed on a capillary column (Acclaim PepMap100, C18, 3 µm, 100 Å, 75 µm i.d.×25cm, Thermo Scientific) at an eluent flow rate of 300 nL/min using a linear gradient of 3–25% B after 53 min with a further increase to 80% B after 80 min. Mobile phase A contained 0.1% formic acid in water, and mobile phase B contained 0.1% formic acid in acetonitrile. Mass spectra were acquired in a data- dependent mode with one MS survey scan with a resolution of 60,000 (Orbitrap Elite) and MS/MS scans of the 15 most intense precursor ions in the linear trap quadrupole were used. Identification and SILAC quantification were performed using MaxQuant software (version 1.4.1.1) with the Uniprot human protein database (July 2013). Proteins were filtered with the following criteria: i) not identified only by site, reverse, or known contaminant (MaxQuant), ii) at least one unique peptide, and iii) at least two razor + unique peptides. The unstimulated Jurkat ABE enrichment we published earlier31 (n=6) was re-analyzed using the following new procedure, allowing direct comparison to the stimulated Jurkat ABE dataset described here (n=4). First, missing values were imputed with the median normalized heavy/light ratio of the specific LC-MS/MS run – in many cases this was very close to 1.0 (representing the background). H/L ratios were then transformed to log2 values and the mean H/L ratio and one-sample t-test p values were calculated. Volcano plots were generated plotting the log2 fold-change to the –log10(p) value, giving rise to a pool of high-confidence palmitoylated proteins. Enriched pools were analyzed based on three criteria, described previously31: i) previous reports in other palmitome studies, ii) predicted palmitoylation sites (CSS-Palm 4.0), and iii) predicted TMDs (TMHMM 2.0)33,34.

On-Plate Palmitoylation Assay (OPPA)

Purified targets (VAMP7, LAT) were diluted to 1 µM in buffer containing 50 mM NaH2PO4 (pH 6.5), 500 mM NaCl, 1 mM TCEP and 0.08% DDM. For each time point (0, 5, 10 and 20 min), the diluted target was divided into 12 wells (50 µl each) of a 96-well Ni-NTA-coated plate (Pierce) and allowed to bind for 1 hour. Palmitic acid alkyne (PAA)-CoA was generated by preparing 50 mM Tris (pH 8.1), 14 mM MgCl2, 0.08% DDM, 0.1 mM EDTA, 1.6 mM ATP (Roth), 400 µM coenzyme A (Sigma), 40 µM PAA (Avanti), 0.05 UN/ml acyl-coenzyme A synthetase (Sigma) and 1% BSA (Sigma) and incubating at 37 ˚C for 1 hour. This was diluted two-fold (yielding [PAA-CoA] ~ 20 µM) with 0.3-2 µM purified DHHC PAT (or 1 µM BSA for negative control) with 150 mM

NaH2PO4 (pH 6.5), 300 mM NaCl and 0.08% DDM. Ni-NTA plates were washed 4 times with PBS + 0.05% 8 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Tween-20 (Sigma-Aldrich) (PBST) using a plate washer (Tecan) and 100 µl of the enzyme mixture was added. Reactions were carried out at 37 ˚C with shaking for designated time points, and the reaction was quenched by washing with buffer containing 50 mM NaH2PO4 (pH 6.5), 300 mM NaCl, 40 mM NEM and 0.1% Triton X-100. After the complete time course, plates were washed 4 times with PBST and click chemistry was performed by the addition of 50 µl buffer containing 50 mM NaH2PO4 (pH 6.5), 300 mM NaCl, and 1% Triton X-100. Click chemistry reagents were added as described previously58; briefly, to each 50 µl, the following reagents were added in this order, with vortexing between each step: 1 µl 1 mM biotin-azide (Thermo Fisher) in DMSO (Sigma), 1 µl 50 mM TCEP, 3 µl 16 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (Sigma-Aldrich) prepared in 20% DMSO and 80% t-butanol (Sigma), and 1 µl 50 mM CuSO4 (Sigma). Click chemistry was performed at 37 ˚C for 3 hours with shaking. After washing the plate 6 times with PBST, palmitoylated targets were eluted with

100 µl buffer containing 50 mM NaH2PO4 (pH 6.5), 300 mM NaCl, 1% Triton X-100, 1 mM EDTA and 200 mM imidazole (Roth) for 30 min at 37 ˚C. Eluates were transferred to high-binding plates (Greiner) coated for 3 hr at room temperature with monoclonal anti-FLAG (Sigma-Aldrich) and blocked for 2 hr at room temperature with 3% BSA and allowed to bind overnight at 4 ˚C with shaking. Plates were then washed 6 times with PBST and buffer containing PBS and 0.2 µg/ml Eu3+-streptavidin (PerkinElmer) and incubated at room temperature for 1 hour. After washing 8 times with PBST, fluorescence was measured in 120 µl enhancer solution (15 µM beta- naphthoyltrifluoroacetone (Sigma), 50 µM tri-n-cotylphosphine oxide (Sigma), 6.8 mM potassium hydrogen phthalate (Roth), 100 mM acetic acid (Roth) and 0.1% Triton X-100) using a plate reader (Victor3V, PerkinElmer) with excitation and emission wavelengths of 340 nm and 614 nm, respectively. All measured fluorescence values were normalized to both the t=0 fluorescence value and the µg of protein (DHHC PAT or BSA) used in the reaction, and steady-state rates were calculated over the 20-minute reaction time.

Immunofluorescence Coverslips & dishes preparation. 12mmø coverslips (VWR, 631-0666) were pre-coated with 0.02% of poly-L- for 20 min at room temperature and were washed 3 times in water before being dried and kept for maximum 2 days. 100,000 cells were incubated on coverslips for 30 min in PBS. Fixation. Cells were fixed with 4% paraformaldehyde (Life Technologies, FB002) for 15 min at room temperature, washed once in PBS and excess of paraformaldehyde was quenched for 10 min with PBS 10 mM glycine (Thermo Fisher Scientific, G8898). Coverslips were kept at 4°C in PBS until permeabilization and staining. Staining. Cells were permeabilized for 30 min at room temperature with PBS + 0.2% Bovine Serum Albumin (BSA, Euromedex, 04-100-812) and 0.05% Saponin (Sigma-Aldrich, S4521). Cells were then incubated for 1 hour at room temperature with primary antibody, then washed 3 times with PBS 0.2% BSA 0.05% Saponin and incubated protected from light for 20 min in the same buffer with spun secondary antibodies. After washing once with PBS BSA Saponin, and once with PBS, coverslips were soaked three times in PBS, three times in water, and mounted on slides. Mounting. For regular confocal microscopy, coverslips were mounted with 4-6 µL Fluoromount G (SouthernBiotech, 0100-01) on slides (KNITTEL Starfrost) and dried overnight protected from light before microscope acquisition.

9 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Microscope: Images were acquired with a Leica DmI8 inverted microscope equipped with an SP8 confocal unit using either a 40X (1.35NA) or 63X (1.4NA) objective. Single plane images or Z-stack of images were acquired (pixel size around 60 nm). Analysis of VAMP7 colocalization with Giantin. Z-stack (0.5 µm) images of similarly dimensioned Jurkat cells were chosen. In this z-stack, an ROI surrounding the Golgi was defined based on Giantin staining. Within each ROI, masks based on both Giantin and VAMP7 stainings were created by thresholding. Automatic colocalization assays were performed with Mander’s overlap coefficient, using the JACoP plugin for ImageJ64. Antibodies: Anti-Flag (1/100) was from Sigma Aldrich (F3165). Anti-Giantin (1/100) was produced by the recombinant antibody platform of the Institut Curie, Paris, France. Anti–rabbit Ig Alexa Fluor 488 (1/200) and anti–mouse Ig Alexa Fluor 568 (1/200) antibodies were from Thermo Fisher Scientific (A11034 and A11004 respectively).

Acknowledgements Special thanks to Frank Kuppler, Ellie Fox, Michael Schümann, and Heike Stephanowitz for technical support. Special thanks to Andrew Peden (The University of Sheffield, UK) for gifting us the anti-VAMP7 antibodies. CF and BB are supported by the DFG (TRR186, Project A05). CH is supported by funds from Institut Curie, Institut National de la Santé et de la Recherche Médicale (INSERM) and ANR (ANR-10-IDEX-0001-02 PSL*, and ANR- 11-LABX-0043).

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Suppl. Table 1 Proteins enriched before and 10 min. after T-cell stimulation

Unstim. Costim. # Pred. log2(fol Unstim. log2(fol Costim. # Lit. # Pred. Gene Accession Palm. d p d p Refs TMDs Sites change) change) B4DDG0;B4DEA5;B4DEB8;B4E 2.01113 0.02028 1.40322 0.20272 TSPAN7 1 6 4 171;P41732 955 114 843 589 2.25172 0.01662 1.85336 0.00318 CANX B4DGP8;B4E2T8;P27824 9 2 1 33 333 5561 279

B4DTE8;C9J0H3;C9J7K9;C9J9P 2.06638 0.00251 2.05039 3.06E- PLSCR1 7 11 0 4;C9JE06;H7C5I5;O15162 636 417 3113 05

2.34024 0.01452 1.56215 0.00562 STMN3 B7Z5G4;Q9NZ72 0 2 0 883 18 7774 693 B7Z5Z2;E9PK85;P62070;P6207 1.78366 0.00616 1.67748 0.12188 RRAS2 8 2 0 0-2;P62070-3 469 599 9209 016 1.42055 0.04145 0.20909 0.24687 EMB B7Z902;D6RDX7;Q6PCB8 0 1 1 727 358 8056 133 E9PB14;H0YD97;O00330;O00 2.44926 0.00136 2.18065 0.01830 PDHX 2 0 0 330-2;O00330-3 863 65 7459 675

E9PFA8;H0Y5K5;H0Y621;H7BX 2.57182 0.00654 1.44169 0.04642 ERGIC3 I5;Q9Y282;Q9Y282-2;Q9Y282- 6 1 2 327 162 1104 445 3

E9PN10;E9PND3;O00559;Q5Y 2.28844 0.03804 1.45023 0.04307 EBAG9;PDAF 4 1 0 8C7;E9PJ38;E9PJ40 683 538 6114 747 2.50368 0.00152 2.32863 0.00266 CD1C H0Y6Y6;P29017 0 0 1 095 703 8675 696 I3L161;I3L3X5;I3L4F5;I3NI29; 1.78957 0.00425 2.32253 0.01509 PLSCR3 6 6 0 Q9NRY6 811 765 8859 676

J3KNF8;D6RFH4;H3BUX2;O43 1.54342 0.03510 0.88393 0.00988 CYB5B 6 0 1 169 443 041 0117 124 1.37867 0.00206 1.32363 0.02338 ALDH6A1 J3KNU8;Q02252;G3V4Z4 1 2 1 031 943 4359 239

E9PQ58;J3KPN1;Q96BS2;Q96B 2.36108 0.01840 2.18136 0.00030 TESC 2 0 0 S2-2;Q96BS2-3;F5H1Y5 119 38 3928 648

1.87756 0.04055 1.58271 0.10693 CD7 J3QLM0;P09564;J3QLC7 1 0 1 996 534 3834 439

H3BM38;H3BNE1;H3BP15;H3 2.60906 0.00169 2.41556 0.01525 SNAP23 BPJ0;H3BQY9;H3BR18;H3BV9 11 5 0 241 896 8364 215 9;O00161;O00161-2

1.51662 0.04152 0.41181 0.16860 ADAM10 O14672 6 4 1 538 201 9875 091 2.02664 0.00565 2.49256 0.00312 SCAMP3 O14828;O14828-2 9 1 4 991 556 16 787 B4DET3;K7EQ23;O15118;F5GZ 1.88752 0.00761 0.90451 0.12672 NPC1 4 11 12 D4 447 025 3216 085

H3BN93;H3BPL9;H3BUB6;H3B 2.00985 0.00818 1.98789 0.00207 SCAMP2 8 0 4 V04;O15127 332 157 8107 22 1.82960 0.03597 1.15385 0.15253 STX7 O15400;O15400-2 3 0 1 361 913 2444 503

15 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2.27200 0.02877 2.37192 0.00452 TRAPPC3 O43617;O43617-2;A6NKE1 8 1 0 673 727 8246 526 3.12266 0.01116 2.21631 0.01889 VAMP4 O75379;O75379-2 4 0 1 383 656 3931 97 1.81215 0.00089 2.73491 0.00091 HRAS P01112;P01112-2 10 2 0 795 66 7125 377

F6U0H7;P01889;P30460;P304 75;P30479;P30480;P30486;Q0 4826;Q29836;Q31610;Q9536 2.25299 0.01387 2.92727 0.00284 HLA-B 5;H0Y4L0;H0Y5W5;P10319;P1 4 10 1 112 229 4958 882 8464;P30464;P30483;P30484; P30487;P30488;P30490;P304 91;P30498;P30685

1.27184 0.04346 1.93865 0.00146 TFRC F5H6B1;G3V0E5;P02786 8 0 1 169 104 8067 596 E9PMT5;H0YE18;P04234;P042 2.32739 0.03133 1.84118 0.00324 CD3D 3 0 0 34-2;A8MVP6 882 549 8817 459

E9PJ92;P06239;P06239- 1.12858 0.01893 1.31367 0.00678 LCK 5 4 0 2;P06239-3;Q573B4;E9PAP0 817 449 819 854

C9JDE9;H7C131;P09110;P091 1.02871 0.00606 1.45082 0.00852 ACAA1 2 0 0 10-2;G5E935 458 781 4748 406 1.63263 0.00724 2.03361 0.00148 RRAS P10301 7 1 0 507 979 7774 504 E9PEJ4;F5H7M3;H0YDD4;P10 1.74415 0.00434 1.68192 0.01632 DLAT 2 2 0 515 836 651 5145 809 1.22280 0.03211 1.53215 0.00911 DBT P11182;Q5VVL7 0 0 0 118 251 3612 031

A8MQT7;H7C2F2;P14209;P14 2.64165 0.01791 1.05034 0.27191 CD99 4 0 2 209-2;P14209-3;A6NIW1 237 371 3019 704

P15151;P15151-2;P15151- 2.82650 0.00248 1.98805 0.01887 PVR 5 0 1 3;P15151-4 499 18 0635 505

P16284;P16284-2;P16284- 1.99049 0.02028 1.12437 0.11612 PECAM1 3;P16284-4;P16284-5;P16284- 4 2 1 366 736 1288 303 6

F5GXE0;F5GXU0;F5H883;P206 2.68618 0.00828 2.06832 0.00153 M6PR 4 1 1 45 707 383 7216 691

P23229;P23229-2;P23229- 1.14350 0.04446 0.20909 0.24687 ITGA6 3;P23229-4;P23229-5;P23229- 3 6 1 104 927 8056 133 6;P23229-7;P23229-9

1.48493 0.00760 1.65821 0.00419 ACAT1 P24752;E9PKF3 2 1 0 077 669 064 804 1.93213 0.02729 0.75185 0.16620 FAS P25445;P25445-6 1 0 0 818 855 2595 912 E9PJC7;P27701;P27701- 1.59383 0.00544 1.59882 0.01050 CD82 7 4 3 2;E9PC70 416 347 7521 883 2.33056 0.00050 0.92248 0.25459 CD38 P28907;H0Y950 3 4 1 519 841 3794 498

16 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

P30504;A2AEA2;P10321;A2AE 2.11980 0.01397 1.55341 0.07526 HLA-C 4 0 1 A4;F5GXA6 81 787 6267 089 1.52651 0.02320 2.19673 0.00337 SLC7A1 P30825 4 2 14 508 005 1373 919 K7EJ20;K7EKX7;K7ENB4;K7ER 1.00209 0.04709 0.69980 0.01961 GPX4 1 2 0 P4;P36969;P36969-2 978 84 2278 693

B4DKB2;P42892;P42892- 1.77635 0.00166 0.21014 0.66772 ECE1 5 2 1 2;P42892-3;P42892-4 042 131 624 525 2.55823 0.00356 2.19652 0.00147 GNAQ P50148 10 2 0 299 05 5936 265 2.44141 0.00029 2.04364 0.00493 RAP2B P61225 10 1 0 831 363 1879 394 1.41958 0.01573 2.81774 0.00092 REEP5 E2QRG8;Q00765 5 1 2 669 059 3268 179 1.73225 0.04932 0.22235 0.50385 CKAP4 Q07065 5 0 1 073 325 1852 91 2.84617 0.00125 1.76218 0.04607 BST2 Q10589;Q10589-2 3 1 1 683 307 0381 302

Q13308;Q13308-2;Q13308- 3;Q13308-4;Q13308- 1.58824 0.02581 0.95417 0.02811 PTK7 3 3 1 5;Q13308- 661 234 4108 198 6;Q86X91;E9PFZ5;H0Y8F1

1.68597 0.02646 2.31942 0.00738 TMED1 K7EIN4;K7EQ63;Q13445 3 0 1 343 665 215 703 1.55534 0.00670 1.02718 0.16951 SCARB2 Q14108;Q14108-2;E7EM68 7 2 2 049 921 3591 739 1.93366 0.00511 1.85832 0.04278 GNA13 F5H1G8;Q14344 10 1 0 229 495 4489 863

M0QXM4;Q15758;Q15758- 2.20476 0.00775 2.55121 0.00295 SLC1A5 9 2 6 2;Q15758-3;B4DWS4;E9PC01 587 502 8874 182

2.27672 0.01662 2.09279 0.06352 VAMP3 Q15836 9 0 1 137 37 307 477 1.89850 0.01705 1.74629 0.00912 ABHD17B Q5VST6;Q5VST6-2 7 6 0 555 32 8039 825 1.79725 0.00212 2.12893 0.01426 SPRYD7 Q5W111;Q5W111-2 3 4 0 435 522 8794 936 2.09026 0.00782 1.78697 0.04161 AP1AR Q63HQ0;Q63HQ0-2 4 2 0 337 167 4152 713 1.96892 0.01405 2.51923 0.01285 MBLAC2 Q68D91;Q68D91-2 5 0 0 624 721 7581 09 F5GX19;F5H3Y3;F5H479;H0YF 2.78042 0.00409 2.93212 0.00350 LAMTOR1 8 2 0 I1;Q6IAA8 824 654 3182 293

1.16714 0.01374 1.85739 0.00254 C8orf82 H0YF29;Q6P1X6;Q6P1X6-2 3 1 0 65 503 3538 588

Q7Z5G4;Q7Z5G4- 3.46812 0.00445 3.22704 0.00217 GOLGA7 6 0 0 3;J3KN76;J3KQ24 661 687 9799 065 2.17875 0.01420 1.53683 0.01106 MTDH E5RJU9;Q86UE4 9 0 1 641 171 1027 392 1.71066 0.02448 1.11368 0.10064 STX12 B1AJQ6;Q86Y82 8 0 1 273 5 6258 077 1.53525 0.04913 0.42537 0.32317 TMEM192 Q8IY95;Q8IY95-2 1 0 4 977 502 9503 932 1.48026 0.03373 0.52697 0.20278 KIAA2013 Q8IYS2;Q8IYS2-2 7 0 2 727 831 5396 664

17 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

C9JAG4;C9JFU1;C9JYV9;Q8N5 2.03865 0.02495 2.22862 0.04322 MREG 4 6 0 65;Q8N565-2 457 843 1622 007

Q8ND76;Q8ND76-2;Q8ND76- 2.11178 0.01119 2.21556 0.00175 CCNY 5 2 0 3;B7Z8E4 182 807 7467 192 1.50933 0.04017 1.92754 0.24127 SVIP Q8NHG7 4 2 0 235 928 3561 28 1.65023 0.02838 1.20867 0.09683 PI4K2B G5E9Z4;Q8TCG2 4 4 0 886 807 5382 168

E7ENA9;F6Y097;Q92542;Q925 1.56372 0.00441 0.96912 0.04357 NCSTN 2 1 1 42-2;H0Y3Z4;H0Y6T7;Q5T205 824 701 5391 303

1.88707 0.03229 1.49907 0.03156 SEMA4D E9PFD9;Q92854;Q92854-2 3 3 1 145 803 0378 584

C9J8Z4;Q969P0;Q969P0- 1.33544 0.02295 1.71210 0.01034 IGSF8 3 3 1 2;Q969P0-3 321 456 187 985 1.81388 0.01755 0.84339 0.16333 TMX3 Q96JJ7;Q96JJ7-2 7 2 1 281 314 6829 816

B4DIP2;Q96RT1;Q96RT1- 2;Q96RT1-3;Q96RT1- 1.83060 0.00261 1.07941 0.10415 ERBB2IP 4;Q96RT1-5;Q96RT1- 7 4 0 13 196 8942 739 6;Q96RT1-7;Q96RT1- 8;Q96RT1-9;E9PCR8

1.93183 0.01151 0.80260 0.02969 SORT1 Q99523;Q99523-2;B4DWI3 3 0 1 09 239 4556 959 3.28343 0.00674 3.33262 0.01665 MNF1 Q5TAQ0;Q9BRT2;V5IRT4 2 1 0 628 742 2664 253 2.07183 0.00691 1.79720 0.03931 LRRC1 Q9BTT6;Q9BTT6-2 5 3 0 912 471 3483 393 2.06689 0.00788 1.68898 0.04231 PI4K2A E9PAM4;Q9BTU6 9 4 0 104 415 0857 594 1.78124 0.00643 1.89082 0.00173 ACAT2 B7Z233;Q9BWD1 2 0 0 516 934 3497 922 H7C1Z7;H7C270;Q9GZU7;Q9G 1.86343 0.01713 2.40765 0.00090 CTDSP1 4 2 0 ZU7-2;Q9GZU7-3 676 732 2335 958 2.04842 0.00871 1.68871 0.02009 TMX4 Q9H1E5 6 1 1 633 556 8909 764 2.41523 0.00773 2.36674 0.02234 TMX1 Q9H3N1 8 0 3 746 626 5076 855 1.89999 0.00340 0.20909 0.24687 DNAJC5 Q9H3Z4;Q9H3Z4-2 8 14 1 039 597 8056 133 1.98668 0.03902 2.60311 0.00377 LIME1 Q9H400 1 3 1 416 498 0399 166 1.78165 0.04824 0.70827 0.03456 TMEM206 Q9H813;Q9H813-2 2 0 1 714 694 4631 783 1.87003 0.00906 1.12094 0.10417 PAG1 Q9NWQ8 1 1 1 008 192 7587 339 2.62207 0.00617 2.70863 0.01871 ARL15 Q9NXU5 5 3 0 713 192 1521 234 1.63389 0.01921 1.46059 0.03441 LNPEP Q9UIQ6;Q9UIQ6-2;Q9UIQ6-3 7 0 1 535 66 7346 924 1.93169 0.00400 1.75728 0.00982 STX8 Q9UNK0;K7EQB1;I3L305 5 1 1 867 296 811 368

E2QRE5;Q9Y228;Q9Y228- 1.85112 0.01162 1.51732 0.05290 TRAF3IP3 4 1 1 2;Q9Y228-3;B1AJU2;C9JBA0 461 809 6134 546

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2.22545 0.00051 2.13011 0.00634 RAP2C;RAP2A F6U784;Q9Y3L5 10 1 0 979 365 4813 896 2.25541 0.02110 2.02578 0.05883 AUP1 Q9Y679;Q9Y679-2;Q9Y679-3 5 2 1 961 216 7434 228

E7ET49;Q8NCG7;Q8NCG7- 1.26418 0.03128 1.60233 0.01105 DAGLB 6 13 3 2;Q8NCG7-4 91 35 605 365

B4DGG1;H7C2U8;Q9H0X4;Q9 1.42339 0.02376 1.18233 0.07167 ITFG3 1 0 1 H0X4-2 586 018 4102 113

Q9HCP0;Q9HCP0- 1.66946 0.01727 2.20173 0.00684 CSNK1G1 3 3 0 2;U3KQA5;U3KQB3;U3KQN9 055 325 0433 368

C9J2G3;C9JQQ2;Q9NWU1;Q9 1.37200 0.02223 1.02424 0.08319 OXSM 4 2 0 NWU1-2 193 416 0546 646

B4DKH7;B4DPY5;J3KTN6;J3QL 0.72296 0.13810 1.79709 0.00282 SNX11 0 4 0 V8;J3QRB9;Q9Y5W9 367 234 7788 784

0.33773 0.71499 1.79707 0.01623 STOM B4E2V5;P27105;P27105-2 8 1 1 636 862 2727 706 1.71820 0.05123 1.86063 0.01718 TNFRSF8 D3YTD8;P28908 1 2 1 482 811 9392 656 1.39976 0.05986 1.60629 0.00311 SCAMP1 O15126;O15126-2;U3KQ30 8 1 4 067 788 9608 923

B7WPI0;C7C5T6;C7C5T7;G5E9 2.06348 0.05536 1.14281 0.03075 LAT K3;O43561;O43561- 3 2 1 859 856 3833 012 2;H3BQD8 2.10188 0.09102 1.55791 0.03435 ITM2A O43736;O43736-2 2 0 1 269 099 0605 444 0.76369 0.23592 1.95508 0.02138 STX11 O75558 5 5 0 501 443 636 748 0.85553 0.03820 2.22204 0.00080 RP2 O75695 2 2 0 755 815 9727 538 1.35304 0.09787 1.86016 0.02576 NRAS P01111 9 0 0 179 449 356 803 1.22306 0.05470 1.51568 0.02473 GPX1 P07203 1 2 0 625 53 5222 32 0.66423 0.15836 1.89288 0.00281 GNAI3 P08754 10 1 0 621 735 9465 359

H0YF61;P22307;P22307- 2;P22307-4;P22307-6;P22307- 0.41918 0.20968 1.33922 0.00607 SCP2 7;P22307- 2 5 0 755 103 5021 81 8;A6NM69;B4DHP6;C9JC79;E1 B6W5

0.60179 0.02244 1.31712 0.00225 ALDH1B1 P30837 2 3 0 253 929 7576 776 0.92290 0.08053 1.88737 0.00084 VAMP7 P51809;P51809-2;P51809-3 7 1 1 231 258 4387 773

P63092;P63092-2;P63092- 0.82217 0.01406 1.39908 0.02310 GNAS 3;P63092-4;Q5JWF2;Q5JWF2- 5 2 0 381 068 2041 608 2;S4R3E3;A6NI00

1.46256 0.05194 1.23667 0.04719 SCRIB Q14160;Q14160-2;Q14160-3 6 2 0 278 783 1939 166 0.88529 0.01936 1.86186 0.00116 MLEC F5H1S8;Q14165 9 0 1 344 323 0164 099

19 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1.23254 0.16008 1.41696 0.00821 CNST Q6PJW8;Q6PJW8-2;Q6PJW8-3 1 1 1 922 345 64 51 0.50093 0.00171 1.08299 0.02190 MCAT Q8IVS2 0 0 0 071 026 3965 818 G3V3Z5;G3V583;Q8N128;Q8N 1.59536 0.05196 2.45095 0.01246 FAM177A1 2 0 0 128-2 037 52 158 697 0.99594 0.00991 1.91098 0.00211 ERGIC1 Q969X5;Q969X5-2 2 2 1 314 418 7753 48 K7EKW4;K7ENV7;Q96AB3;Q9 0.35324 0.22848 1.59246 0.00387 ISOC2 0 1 0 6AB3-2;Q96AB3-3 72 477 1706 52 1.38841 0.06129 2.20317 0.00133 DESI2 Q9BSY9;Q9BSY9-2 2 0 0 244 207 996 655 0.85168 0.00085 1.39352 0.01289 FAM49B E5RI16;Q9NUQ9;Q9NW21 8 2 0 277 867 0601 23 0.85185 0.12745 1.14363 0.03229 SLC5A6 Q9Y289 4 2 14 021 011 9019 978

B3KW46;B4DZ71;B7ZKQ9;F5H 4X0;F8W8N0;Q8WTV0;Q8WT 0.73680 0.11187 1.62979 0.00279 SCARB1 5 2 2 V0-2;Q8WTV0-3;Q8WTV0- 02 271 741 175 4;Q8WTV0-5

1.11500 0.14250 1.74678 0.00351 GNA15 P30679 3 3 0 187 121 995 838 0.77892 0.13505 1.46973 0.01375 CSNK1G2 K7ESB6;P78368 3 3 0 34 561 6331 202 A6NEP9;H7BXY6;Q8NG11;Q8 1.36704 0.09826 2.34908 0.00105 TSPAN14 3 4 1 NG11-2;Q8NG11-3 746 294 6708 765

0.46543 0.08194 1.82971 0.01902 NAT14 M0R1E3;Q8WUY8;M0QZH3 0 0 1 415 934 1371 646

0.75254 0.19569 1.06508 0.02952 CHST11 F8VXK3;Q9NPF2;Q9NPF2-2 1 3 1 062 366 3361 848

P06241;P06241-2;P06241- 0.87742 0.07045 1.01476 0.01294 FYN 3;Q5R3A8;E5RFS5;E5RGT0;E5 6 5 0 116 254 9118 14 RH71;E5RIX5;B3KPS6

0.61238 0.01971 1.44991 0.00016 RALA H7C3P7;P11233;C9JPE8 6 2 0 472 696 2626 727 0.67574 0.28116 1.65960 0.04525 SLC30A1 Q9Y6M5 0 3 6 27 244 5285 969 0.42162 0.25246 1.27098 0.01614 FNDC3A G5E9X3;Q9Y2H6;Q9Y2H6-2 2 0 1 613 438 6869 71

A2AB09;A2AB10;A2AB12;B0V 1.00459 0.08195 1.55982 0.01212 FLOT1 109;B0V111;B4DVY7;O75955; 10 2 0 554 335 289 381 F8VSI6

F8VPA6;H0YI50;Q96RQ1;F8W 0.76513 0.13570 1.47505 0.01088 ERGIC2 2 1 0 0R1 754 15 7844 79

B0UXB6;B3KNX9;O95870;O95 0.63145 0.17714 1.48271 0.02409 ABHD16A 5 3 2 870-2 647 745 0847 631

Q3SXY8;Q3SXY8-2;Q3SXY8- 0.53943 0.30106 1.40418 0.01637 ARL13B 1 2 0 3;G3V1S8 282 527 8346 17

E9PJN0;H7C5A7;O14734;E9PI 0.25842 0.13069 1.21164 0.02213 ACOT8 1 2 0 S4;E9PMC4;Q9BR14;E9PRD4 179 237 1176 307

20 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

0.36324 0.23240 1.21546 0.01540 NAT1 F5H5R8;P18440 1 0 0 027 497 2463 887 0.21824 0.63914 1.23782 0.00776 PAFAH2 Q99487;Q5SY00 1 0 0 36 582 0847 988 E7EMK3;J3QLD9;Q14254;K7E 1.03883 0.13955 1.33028 0.02710 FLOT2 9 3 0 KW9 305 775 0528 432 0.71199 0.33312 1.21009 0.00829 VANGL1 Q8TAA9;Q8TAA9-2 2 0 4 456 105 163 22 B4E040;C9J6B1;C9JQB3;P1123 0.94614 0.05546 2.00812 0.00371 RALB 4 2 0 4;Q6ZS74;C9JYR1 879 476 8932 793 0.07421 0.36070 1.27485 0.01899 HSD17B8 Q92506 0 1 0 329 022 5828 502 Q9H6X2;Q9H6X2-2;Q9H6X2- 0.81211 0.25358 1.23843 0.01011 ANTXR1 4 0 1 3;Q9H6X2-4 628 608 5563 6 0.50086 0.21097 1.37833 0.00050 SLC1A4 P43007;P43007-2 2 0 9 828 511 5147 182

G3V267;G3V4T6;G3V5T0;O43 - 0.75352 1.35927 0.02860 GSTZ1 708;O43708-2;O43708- 0.03324 0 0 0 227 8191 855 3;A6NED0;G3V3B9 713

0.11350 0.34482 1.20251 0.01853 HMGCL B1AK13;P35914;P35914-2 0 0 0 167 914 086 95 0.40725 0.34248 1.67119 0.02204 GNAO1 P09471;P09471-2 4 1 0 095 431 531 512 0.41649 0.37379 1.96963 0.01439 C9orf91 Q5VZI3;Q5VZI3-2;Q5VZI3-3 0 3 2 939 333 6987 981 0.07164 0.46487 1.03319 0.00012 KRAS P01116;P01116-2 4 0 0 405 866 8072 044 - Q8NE86;Q8NE86-3;Q8NE86- 0.76091 1.12350 0.01641 MCU 0.04431 2 0 2 2;S4R468 485 2562 4 739 - 0.75580 1.03981 0.00287 ZDHHC3 F8W6M3;Q9NYG2;Q9NYG2-2 0.01328 2 3 4 924 5216 018 13 B4DDM2;F5H7Y9;Q8TB61;Q8T 0.78153 0.15815 1.83032 0.00087 SLC35B2 5 1 7 B61-2;Q8TB61-3 582 232 0405 649

E7ES96;P49768;P49768- 2;P49768-3;P49768-5;P49768- 0.17119 0.41756 1.28531 0.01327 PSEN1 6;P49768- 5 0 8 673 108 781 083 7;G3V2G7;G3V3Z0;G3V499;P4 9768-4

Q96CB9;Q96CB9- 0.09518 0.47874 1.20107 9.32E- NSUN4 4;M0R1K5;Q96CB9- 0 0 0 292 675 0943 05 2;B4DHA4;Q96CB9-3

0.59657 0.37752 1.94705 0.02342 ZDHHC18 Q9NUE0;B4DQ84;H0YEN1 3 2 4 371 488 2819 713 0.39580 0.37592 1.92706 0.00024 LIPT2 A6NK58 0 0 0 855 866 7631 427 0.70384 0.39351 1.48829 0.01243 SERINC1 Q9NRX5 4 6 11 619 487 3246 243 0.48495 0.38100 1.04899 0.02747 SLC9A6 Q92581;Q92581-2;Q92581-3 2 2 11 458 128 2629 108 0.49029 0.43473 1.29029 0.01367 ATP11B H7C4W6;Q9Y2G3 4 4 8 404 166 0032 196

21 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

H3BTQ3;P41440;P41440- 0.60127 0.16088 1.31127 0.04689 SLC19A1 3 0 10 2;P41440-3 418 957 506 647 0.71995 0.12287 1.65276 0.00950 RHBDD2 Q6NTF9 3 0 5 523 565 4543 578 0.37036 0.38687 1.37675 0.01806 PLCXD1 C9JP92;Q9NUJ7 0 0 0 826 81 1589 04 - 0.75580 1.73441 0.02693 CTDSP2 H0YI12;O14595;F8VYL0 0.01328 2 2 0 924 8165 23 13 0.16197 0.42523 1.90673 0.00173 TMPPE Q6ZT21;Q6ZT21-2 2 0 5 153 163 8539 242 - E5RFT4;E5RIY0;Q8N4L2;E5RIP 0.47479 1.87799 0.02159 TMEM55A 0.08487 1 5 2 9;E5RJC2 362 1 247 484 0.36355 0.17440 1.10815 0.01417 ZDHHC6 Q9H6R6;Q9H6R6-2 1 6 4 781 261 6078 619 - 0.96286 1.35100 0.02201 SLC38A1 F8VX04;Q9H2H9 0.00209 0 0 11 382 1155 907 62 C9JWM2;K7EJJ4;Q969E2;Q96 0.02144 0.24227 1.77339 0.02087 SCAMP4 9E2-2;Q969E2- 3 1 3 896 886 3791 288 3;F8WDW4;H7BZ59 0.25537 0.32131 2.11322 0.00022 RCE1 E9PI08;Q9Y256;E9PKA7 2 0 6 594 494 042 074 - 0.40639 1.04053 0.03260 LCHN;KIAA1147 A4D1U4;C9J7X6 0.18462 0 6 0 768 3671 786 736 0.15699 0.31384 1.42583 0.02555 KIR3DL2 P43630;P43630-2 0 1 1 785 703 636 133 0.08597 0.48318 1.34485 0.01874 GRPEL2 Q8TAA5 0 0 0 592 917 2625 903

D6R9S8;D6RA76;D6RAZ7;D6R C11;D6RCE6;D6RCR8;D6RD48; - D6RDI6;D6RDP6;D6RDY1;D6R 0.33292 1.12352 0.00876 CLDND1 0.25583 4 1 2 FX6;D6RHU6;D6RIU2;H0Y8T9; 919 2085 689 873 J3KQ67;Q9NY35;Q9NY35- 2;D6RHI6

D6R925;D6R9M9;D6R9S7;D6R 9X6;D6RC02;D6RDB0;D6RF72; - D6RHE3;D6RJC0;E7ENY4;Q96 0.75580 1.45062 0.01493 SLC41A3 0.01328 1 0 10 GZ6;Q96GZ6-2;Q96GZ6- 924 7074 075 13 3;Q96GZ6-4;Q96GZ6- 5;Q96GZ6-6;Q96GZ6-7

- 0.75580 1.42174 0.00381 TRGC2;TRGC1 P03986;P0CF51 0.01328 0 6 1 924 1802 972 13

B3KTZ0;B4E2X5;F5GZL8;P0489 - 0.75580 1.88424 0.00541 GNAI2;GNAI1 9;P04899-2;P04899-3;P04899- 0.01328 10 2 0 924 8556 97 4;P63096 13

- H0Y308;Q15561;Q15561- 0.75580 1.16921 0.04728 TEAD4;TEAD2 0.01328 0 0 0 2;Q15562;Q53GI4;Q8NA25 924 2711 847 13

22 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

- 0.75580 2.36493 0.00213 SLC35F6;C2orf18 B4DLH2;Q8N357 0.01328 4 0 10 924 6708 765 13 - 0.75580 2.21941 0.01606 SPPL3 Q8TCT6;Q8TCT6-2;Q8TCT6-3 0.01328 2 0 3 924 6895 056 13 - I3L072;Q9BSJ5;Q9BSJ5- 0.75580 1.47008 0.02351 C17orf80 0.01328 0 4 1 2;Q9BSJ5-3;J3KT60;J3KTJ7 924 1418 217 13

23 bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fig. 1

A B Unstimulated D

C 10-min. Costimulated E

F G TCR signaling Ras signal transduction Cytokine Leukocyte activation production

Metabolism Ubiquitination Synaptic plasticity Vesicle- mediated transport Development Apoptosis

Ion/ transport Regulation of catalysis p-value bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1. Enrichment of Jurkat T-cell palmitome before and after anti-CD28/anti- CD3-costimulation by acyl-biotin exchange. A. Schematic representation of acyl- biotin exchange (ABE) protocol coupled with SILAC for quantitation of palmitoylated proteins via LC-MS/MS. Briefly, Jurkat cells grown in heavy SILAC media (blue) were lysed and free thiols were blocked with NEM. After cleaving palmitoyl groups with HA, previously palmitoylated thiols are biotinylated with HPDP-biotin, allowing for streptavidin enrichment. For control cells grown in light SILAC media (red), HA is omitted, preventing biotinylation. Proteins quantified with heavy/light intensity ratios > 2.0 are considered enriched and palmitoylated. B. Volcano plot showing high-confidence palmitoylated proteins prior to stimulation (n=6, p < 0.05). Proteins found in five or more other palmitome studies are highlighted in orange. C. Volcano plot showing high-confidence palmitoylated proteins 10 minutes after anti-CD28/anti-CD3 costimulation (n=4, p < 0.05). D. Comparison of high- confidence palmitoylated pools before and after stimulation. E. Analysis of palmitoylated proteins compared to unenriched (background) proteins, with three metrics: at least five references in previous palmitome studies, at least one predicted palmitoylation site (CSSPalm 4.0, high cutoff), and at least one predicted transmembrane domain (TMHMM 2.0). F. STRING functional analysis of proteins enriched for palmitoylation 10 minutes after TCR stimulation. A clear cluster of Gα and Ras-related proteins is observed. Several interesting candidates are highlighted, including VAMP7 and DHHC18, which were investigated in more detail in this study. G. Gene Ontology (GO) term enrichment of stimulation-dependent palmitoylation candidates, generated using BiNGO. Cluster size indicates the number of in each node, while color indicates statistical significance of enrichment. bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 2 A D

VAMP7- VAMP7- LAT- WT C183A WT

N C B WT Δ WT WT WT WT WT WT BSA - -Δ - - - BSA - - - - C222A C138A C156A BSA C222A C156A FL -C138A- - -FL - -FL -FL -FL - -FL - - -iso2 FL FL -FL -FL FL - - -iso2 - DHHC18DHHC18 DHHC20 DHHC18DHHC20 DHHC18 DHHC20 DHHC18 DHHC18 DHHC20 DHHC18 DHHC18DHHC18 DHHC18 DHHC20

C bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2. In vitro palmitoylation of VAMP7 by DHHC18 and DHHC20. A. Schematic cartoon of constructs used, with important cysteines highlighted. Cys222 and Cys156 are the active sites of DHHC18 and DHHC20, respectively. Cys183 in VAMP7 is the site of palmitoylation. B. Cartoon model of potential membrane docking of DHHC18 isoform 2 via Cys138. The full-length DHHC18 features the two N-terminal TMDs, securing the DHHC active site to the membrane. The short isoform, lacking these TMDs, may be anchored to the membrane via palmitoylation of Cys138, allowing its active site access to the membrane environment required for its activity. C. Schematic representation of the on-plate palmitoylation assay (OPPA). Briefly, the target (VAMP7, featuring both His6 and FLAG tags) is bound to a Ni-NTA plate. The DHHC PAT is added with clickable PAA-CoA and the reaction is quenched at specific time points using NEM. Biotin is subsequently clicked on to the PAA-modified target, VAMP7 is eluted and then bound to a anti-FLAG plate. Streptavidin-Eu3+ can then be used to quantify palmitoylated VAMP7, and a steady-state slope can be calculated. D. Results of OPPA experiments of VAMP7-WT and VAMP7-C183A, palmitoylated by constructs of DHHC18 and DHHC20 (n ≥ 12, geometric mean +/- SEM shown). bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 3

A DAPI VAMP7-Flag Giantin Merge WT

B

C183A bioRxiv preprint doi: https://doi.org/10.1101/831388; this version posted November 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3. The VAMP7-C183A mutant fails to properly localize to the Golgi. A Confocal images showing the relative localization of VAMP7 (red) labelled with anti-Flag Abs and Giantin (green) in VAMP7-KO Jurkat cells expressing the WT VAMP7-Flagged or the mutant VAMP7C183A-Flagged proteins. Scale bar 5 μm. B Dot plots showing the quantification of the percentage of Giantin (Golgi marker) overlapping with the VAMP7- flagged proteins (Manderson coefficient). Each dot = one cell; horizontal lines = median. ****P < 0.0001 (one-way ANOVA).