Association of NLRP3 with intracellular membranes is dependent on S-acylation.

A) Schematic of the sequences used for GFP tagged minimal NLRP3 constructs expressed transiently in HeLaM cells. B) Representative confocal microscopy images of each construct expressed. Scale bar = 10 µm. C) Quantification of the amount of each mini-construct localised to the Golgi relative to cytosolic signal. N=3. D) Overview of domain architecture of NLRP3. Boxed region indicates region and residues shown below with sequence conservation across 20 species represented by a web-logo plot. E) ChimeraX graphic based on the inactive Cryo-EM structure of NLRP3 (PDB ID: 7PZC) showing a side view of the NLRP3 decamer. Monomers within each pentamer are colour coded blue or green. One monomer from each pentamer is hidden to give a clearer view of helix115-125 (coloured cyan with Cys-130 in pink) and the polybasic region from each monomer (coloured yellow). Ei) Graphic showing the position of residues 95-146 relative to the PYD and NACHT domains. Eii) Zoom in looking along helix115-125. Residues Tryp117, Leu120, Leu121 and Leu124 (labelled) align along a single face of helix115-125. F) Representative confocal microscopy images of HeLaM cells transiently expressing GFP-NLRP3 treated with either 0.5% DMSO or 100 µM 2-BP for 16 hours with or without a 1-hour treatment with 10 µM nigericin and G) quantification of GFP-NLRP3 signal associated with the Golgi from the same experiment. N = 4. Scale bar = 10 µm. H) Representative images of HeLaM cells transiently expressing GFP-NLRP3 or GFP-NLRP3C130S treated with or without 10 µM nigericin for 1 hour. Scale bar = 10 µm. I) Quantification of GFP-NLRP3 or GFP-NLRP3C130S on the Golgi before and after nigericin treatment. N = 4

Overexpression of ZDHHC palmitoyltransferases can alter NLRP3 localisation in a Cys-130 and helix115-125 dependent manner.

A) Quantification of the ratio of GFP-NLRP3 signal on the Golgi relative to that in the cytosol in HeLaM cells transiently transfected with 1 µg of each individual HA-tagged ZDHHC enzyme and GFP-NLRP3. N = 2. B) Representative confocal microscopy images showing the effect of HA-ZDHHC3 and HA-ZDHHC5 on the localisation of GFP-NLRP3. Scale bars = 10 µm. C) Quantification of GFP-NLRP3 or GFP-NLRP3C130S Golgi signal in HeLaM cells transiently transfected with lower amounts (0.25 µg) of each HA-ZDHHC enzyme which altered GFP-NLRP3 localisation in (A). N = 3. **p < 0.01, ***p < 0.001 (Unpaired t test). D) Representative confocal microscopy images of GFP-NLRP3 or GFP-NLRP3C130S expressed with the indicated HA tagged ZDHHC enzyme. HA-ZDHHC9 images demonstrate that not all Golgi localised PATs drive enhanced Golgi recruitment of NLRP3. E) Representative confocal microscopy images of GFP-NLRP3 Golgi recruitment in cells transfected with wild type HA-ZDHHC3 or catalytically inactive HA-ZDHHC3C157S. Scale bar = 10 µm. F) Quantification of GFP-NLRP3 Golgi signal in HeLaM cells co-transfected with HA-ZDHHC3 and catalytically inactive HA-ZDHHC3C157S. N = 3. G) Quantification of GFP-NLRP3 or GFP-NLRP3HF signal on the Golgi in HeLaM cells transiently transfected with 0.25 µg HA-ZDHHC3. N = 3.

NLRP3 localisation to the Golgi is Cys-130 dependent and sensitive to ZDHHC3 levels in THP-1 macrophages.

A) Confocal microscopy images of LPS stimulated THP-1 cells stained with antibodies against NLRP3 and p230. Scale bar = 5 µm. B) THP-1 cells stably transduced with either GFP-NLRP3, GFP-NLRP3C130S, or GFP-NLRP3HF were imaged live by confocal microscopy and scored in C) for the percentage of cells showing a perinuclear enrichment of GFP-NLRP3 signal. N = 3. Scale bar = 10 µm. D) Confocal microscopy images of stable GFP-NLRP3 or GFP-NLRP3C130S THP-1 cells transduced with HA-ZDHHC3 and stained for HA to visualise ZDHHC3 localisation and TGN46. Images are representative of two independent experiments. E) THP-1 cells stably over-expressing HA-ZDHHC3 or HA-ZDHHC3C157S were stained with anti-NLRP3 antibodies to visualise NLRP3 recruitment to the peri-nuclear region. Scale bar = 20 µm. F) Representative live cell confocal microscopy images of LPS primed THP-1 cells stably over-expressing HA-ZDHHC3 or HA-ZDHHC3C157S stimulated with 20 µM nigericin for 2 hours. Dead cells were visualised by propidium iodide staining with cell boundaries visualised through WGA-Alexa647 staining. Scale bar = 20 µm. G) Quantification of the percentage of non-transduced, HA-ZDHHC3 or HA-ZDHHC3C157S THP-1 cells positive for propidium iodide following treatment with 20 µM nigericin for 2 hours. N = 3.

NLRP3 can be S-acylated at Cys-130.

A) Visualisation of S-acylation sites in HeLaM cells transiently expressing GFP-NLRP3 or GFP-NLRP3C130S, labelled by an acyl biotin exchange assay (ABE) with 5-kDa PEG maleimide. Arrowheads indicate non-lipidated NLRP3 (0), singly S-acylated NLRP3 (1) and doubly acylated NLRP3 (2) species. B) Visualisation of acylation sites by 5kDa PEG maleimide ABE in HeLaM cells transiently expressing GFP-NLRP3 or GFP-NLRP3C130S and 0.25 µg HA-ZDHHC3. C) Visualisation of acylation sites by 5kDa PEG maleimide ABE in THP-1 cells stably transduced with GFP-NLRP3 or GFP-NLRP3C130S. Blots are representative of 2 independent experiments. D) Schematic of minimal GFP tagged NLRP3 sequences used and expressed in HeLaM cells to directly assess S-acylation at Cys-130. Mutations introduced are highlighted in green. E) Representative confocal microscopy images of GFP-NLRP395-158, GFP-NLRP3C130S,95-158 or GFP-NLRP3HF,95-158, expressed alone or with 0.25 µg HA-ZDHHC3. N = 3. Scale bars = 10 µm. F) Quantification of GFP-NLRP395-158, GFP-NLRP3C130S,95-158 or GFP-NLRP3HF,95-158 signal associated with the Golgi with or without HA-ZDHHC3. G) Visualization of S-acylation sites in GFP-NLRP395-158 peptides expressed in HeLaM cells by ABE with or without 0.25 µg HA-ZDHHC3.

NLRP3 dynamically associates with the Golgi apparatus.

A) Representative confocal microscopy images of GFP-NLRP3 relative to TGN46 in cells treated for 4 hours with 50 µM PalmostatinB or 0.4% DMSO vehicle control and quantification of GFP-NLRP3 signal associated with the Golgi with or without PalmostatinB treatment. B) Representative images of GFP-NLRP3 in HeLaM cells co-expressed with 0.25 µg APT1-FLAG or 0.25 µg APT2-FLAG with quantification of GFP-NLRP3 signal associated with the Golgi in cells overexpressing APT1-FLAG or APT2-FLAG. Golgi associated GFP-NLRP3 signal is lost in cells expressing APT2-FLAG. C) Representative confocal images of the effect of PalmostatinB treatment on enhanced recruitment of NLRP3 to the Golgi after nigericin treatment and quantification of perinuclear GFP-NLRP3 signal. Cells were pre-treated for 4 hours with 50 µM PalmostatinB or 0.4% DMSO control before a 1-hour stimulation with 10 µM nigericin. D) Representative microscopy images of GFP-NLRP3 and APT1 or APT2-FLAG expressing cells following a 1 hour treatment with 10 µM nigericin and quantification of GFP-NLRP3 signal on the Golgi under the same conditions. E) Representative images of GFP-NLRP3 expressing HeLaM cells before and after photobleaching of Golgi associated NLRP3. F) FRAP recovery curves of GFP-NLRP3 transiently expressed in HeLaM cells treated with vehicle control or 10 µM nigericin for 1 hour. G) Quantification of the mobile and immobile pool of GFP-NLRP3 signal in cells treated with vehicle control or 10 µM nigericin. H) Representative confocal microscopy images of mitochondrial re-routing of FKBP tagged GFP-NLRP3 transiently expressed in HeLaM cells. Cells were treated with either vehicle control or 10 µM nigericin for 1 hour before a 15-minute co-incubation with 500 nM rapamycin prior to fixation. Scale bars in main figure = 10 µm, insets 5 µm. I) Quantification of the amount of GFP-NLRP3 signal associated with the mitochondria following rapamycin treatment in the presence of absence of nigericin. Values are expressed as a percentage of the mitochondrial to cytosolic GFP-NLRP3 ratio seen in vehicle treated control cells.

Nigericin inhibits trafficking through the Golgi apparatus and limits contact of thioesterases with NLRP3.

A) Representative confocal microscopy images of GFP-NLRP3 and APT2S122A-StrepTag following a 1 hour treatment with 10 µM nigericin or vehicle control. Arrowheads point to instances of GFP-NLRP3 signal where APT2S122A is largely absent. B) Linescan analysis of GFP-NLRP3 signal overlap with APT2S122A in control and nigericin treated cells. C) Schematic of EGFP-FM4-hGH secretory reporter system showing the domain architecture of the EGFP-FM4-hGH reporter and position of the reporter within the secretory pathway over time after the addition of rapamycin. D) Representative confocal microscopy images of EGFP-FM4-hGH relative to the cis-Golgi marker STX5 and trans-Golgi marker p230 in HeLa cells treated with 500 nM rapamycin, in the presence or absence of 10 µM nigericin, for 1 hour and E) quantification of EGFP-FM4-hGH signal in the perinuclear area for the same experiment. N = 3. F) Proposed model for S-acylation dependent accumulation of NLRP3 on the Golgi. In the absence of nigericin, NLRP3 shows enrichment on the Golgi due to the balance of acylation and de-acylation provided by the opposing actions of ZDHHC and APT/ABHD enzymes. Access of APT2 to NLRP3 is dependent on anterograde trafficking from cis to trans cisternae remaining intact as APT2 is acylated by ZDHHC3/7, which predominantly localise to the cis-face of the Golgi. G) Nigericin inhibits trafficking through the Golgi complex with exchange of material between medial and trans cisternae impaired. This limits the amount of APT2 able to access NLRP3 leading to its gradual accumulation, likely on trans cisternae and the TGN.

Cysteine-130 is surface exposed in the cryo-EM structure of the inactive NLRP3 complex.

A) View of an individual NLRP3 pentamer from 7PZC and C) an NLRP3 hexamer from 7LFH looking into the core of the decameric complex, where the PYD domains are proposed to be shielded. For 7PZC, the polybasic regions from each monomer are arranged around helix115-125 and Cys-130, with a similar arrangement seen in 7FLH, although helix115-125 and Cys-130 were not resolved in 7FLH. Electrostatic potential of the polybasic regions from each monomer surrounding helix115-125 and Cys-130 mapped onto the structure of the NLRP3 pentamer for B) 7PZC and the NLRP3 hexamer for D) 7LFH.

Dynamics of NLRP3 association with the Golgi post nigericin treatment in HeLaM cells.

A) Representative images of GFP-NLRP3 transiently expressed in HeLaM cells treated with 10 µM nigericin for the indicated times. Scale bar = 10 µm B) Quantification of the Golgi to cytoplasmic GFP-NLRP3 fluorescence for the same experiment. N = 2. C) Mean fluorescence intensity values (MFI) for GFP-NLRP3 in the cytosol and Golgi from cells treated with 10µM nigericin for the indicated times.

Localisation of untagged NLRP3 to the Golgi is S-acylation dependent.

A) Representative confocal microscopy images of HeLaM cells transiently expressing untagged NLRP3 or NLRP3C130S visualised with anti-NLRP3 antibodies. Cells were treated with either 0.5% DMSO or 100 µM 2-BP for 16 hours before treatment with 10 µM nigericin or vehicle for 1 hour. Representative confocal microscopy images of HeLaM cells transiently transfected with B) NLRP3 or C) NLRP3C130S and 0.25 µg of the indicated ZDHHC enzyme. Untagged NLRP3 is sensitive to the same ZDHHC enzymes in a Cys-130 dependent manner as GFP tagged NLRP3.

Hydrophobic residues within helix115-125 are important for localisation of NLRP3 to the Golgi apparatus.

A) Sequence of NLRP3 between residues 110 and 136 and mutations made in the same sequence to disrupt the putative hydrophobic face of helix115-125. B) Heliquest plot of wild type NLRP3 sequence for residues 111-128 and the effect of mutations to residues I113, W117, L120 and L124 (NLRP3HF) on the hydrophobic face. C) Representative images of wild type GFP-NLRP3 and GFP-NLRP3HF transiently expressed in HeLaM cells and treated with 10 µM nigericin for 1hour or left untreated. D) GFP-NLRP3HF is insensitive to ZDHHC overexpression. Representative images of HeLaM cells transfected with GFP-NLRP3HF and 0.25 µg of the indicated HA-ZDHHC enzyme.

Definition of a minimal NLRP3 region required for enrichment at the Golgi apparatus.

A) Schematic of truncations made in full length NLRP3 from the N terminus of the protein. Representative confocal microscopy images of B) GFP-NLRP3Δ95-1036 or C) GFP-NLRP3Δ121-1036 transiently expressed in HeLaM cells, treated with or without 10 µM nigericin for 1 hour. D) Schematic of truncations made in full length NLRP3 from the C terminus of the protein. Representative confocal microscopy images of the E) GFP-NLRP3Δ1-699 or F) GFP-NLRP3Δ1-680 transiently expressed in HeLaM cells, treated with or without 10 µM nigericin for 1 hour. G) Schematic of the minimal Golgi binding truncation mutant NLRP3 Δ95-699 with representative confocal microscopy images of NLRP3 Δ95-699 expressed in HeLaM cells, treated with or without 10 µM nigericin for 1 hour. H) Quantification of the ratio of GFP signal in the perinuclear region over cytoplasmic GFP signal for each of the indicated constructs.

Farnesylation of NLRP3 residues 95-158 permits Cys-130 dependent Golgi association.

A) Representative images of NLRP395-158-CVIM and B) NLRP3C130S,95-158-CVIM transiently expressed in HeLaM cells treated with or without 10 µM nigericin for 1 hour. Boxed region in each image indicates the region shown in higher magnification next to each set of images. C) Quantification of the Golgi association of each construct in untreated cells and in cells treated with 10 µM Nigericin for 1 hour. D) Representative microscopy images showing GFP-NLRP395-158-CVIM co-localisation with untagged NLRP3 in untreated cells and following nigericin stimulation. Cells were stimulated with 10 µM nigericin or vehicle for 1 hour. E) Linescan analysis of GFP-NLRP395-158-CVIM signal with NLRP3 in vehicle treated and nigericin treated cells. GFP-NLRP395-158-CVIM shows a high degree of overlap with NLRP3 under both conditions.

Requirement of the NLRP3 polybasic region for sensitivity to ZDHHC3 and ZDHHC5.

A) Reperesentative images of full length GFP tagged NLRP3 polybasic mutants treated with or without 10 µM nigericin for 1 hour and schematic of mutations made in the polybasic region. Mutated residues are highlighted in orange. B) Representative images of GFP-NLRP3 and GFP-NLRP3PB-1 coexpressed with or without HA-ZDHHC5. Mutation of residues within the polybasic region renders NLRP3 insensitive to ZDHHC5 overexpression. C) Representative confocal microscopy images of GFP-NLRP3, GFP-NLRP3PB-1 and GFP-NLRP3PB-2 transiently expressed in HeLaM cells alone or with HA-ZDHHC3. D) Quantification of GFP-NLRP3, GFP-NLRP3PB-1 and GFP-NLRP3PB-2 Golgi recruitment with and without HA-ZDHHC3. E) Schematic of mutations made in the polybasic region of GFP-NLRP395-158 constructs. Mutated residues are highlighted in orange. F) Representative confocal microscopy images of GFP-NLRP395-158, GFP-NLRP3PB-1,95-158 or GFP-NLRP3PB-2,95-158 expressed in HeLaM cells with or without HA-ZDHHC3. G) Quantification of GFP-NLRP395-158, GFP-NLRP3PB-1,95-158 or GFP-NLRP3PB-2,95-158 signal on the Golgi with and without HA-ZDHHC3. H) Western blots of acyl biotin exchange assay using 5kDa PEG maleimide performed on cell lysates harvested from cells expressing GFP-NLRP395-158, GFP-NLRP3 PB-1,95-158 or GFP-NLRP3PB-2,95-158 with or without HA-ZDHHC3.

PalmostatinB treatment relocalises NLRP3 to the plasma membrane, tubular recycling endosomes and late endosomes.

A) Zoom out of images from Figure 5A showing multiple cells treated with vehicle or 50 µM Palmostatin B for 4 hours. Representative images of GFP-NLRP3 co-localisation with B) Rab8 or C) CD63 in cells treated with vehicle or 50 µM PalmostatinB for 4 hours. Orange arrowheads point to instances of co-localisation between GFP-NLRP3 and the indicated marker in PalmostatinB treated cells.

The effect of APT2 on NLRP3 Golgi enrichment can be reversed.

A) Representative confocal microscopy images and quantification of APT2-StrepTag-FKBP (APT2) signal in the perinuclear region showing re-routing of APT2 onto mitochondrial membranes in the presence of 500 nM rapamycin for 15 minutes and B) Quantification of APT2 signal recruited to the mitochondria relative to cytosolic signal with and without 10 µM nigericin. Nigericin treatment has no significant effect on recruitment of APT2 to the mitochondria. C) Representative images of HeLaM cells transiently expressing GFP-NLRP3 with APT2-StrepTag-FKBP and FRB-mito pre-treated with rapamycin or vehicle for 15 minutes prior to 10 µM nigericin treatment for 1 hour. Removal of overexpressed APT2-StrepTag-FKBP activity restores the enhanced perinuclear NLRP3 signal seen following nigericin treatment to levels seen using APT1, quantified in D). E) Zoom in of boxed regions in C) showing overlap between GFP-NLRP3 and APT2-StrepTag with F) linescan analysis for each set of images.

NLRP3 accumulates on Golgi compartments that are distinct from the cis-Golgi after nigericin treatment.

Representative images of untagged NLRP3 expressed in HeLa cells stably expressing the secretory reporter EGFP-FM4-hGH, treated with 500nM rapamycin for 1 hour in the A) absence or C) presence of nigericin. Following nigericin treatment, NLRP3 shows minimal co-localisation with the cis Golgi marker STX5 and hGH, which is trapped in STX5+ compartments. Line scan analysis of NLRP3 signal overlap with hGH and STX5 in B) vehicle treated control cells or D) cells treated with 10 µM nigericin for 1 hour.

Monensin enhances NLRP3 Golgi recruitment and blocks trafficking through the Golgi.

A) Representative images of HeLaM cells transfected with GFP-NLRP3 and treated with either 10 µM nigericin or 10 µM monensin for 1 hour. Scale bar = 10 µm. B) Quantification of the Golgi to cytoplasmic GFP-NLRP3 ratio following treatment with nigericin or monensin. N=2. C) Representative images of HeLaM cells transfected with an untagged NLRP3 construct and stained with anti-NLRP3 antibodies following treatment with either 10 µM nigericin or 10 µM monensin for 1 hour. Scale bar = 10 µm. D) Representative images of HeLa EGFP-FM4-hGH cells treated with 10 µM nigericin or 10 µM monensin for 1 hour in the presence of rapamycin to solubilise aggregated cargo in the ER. E) Quantification of EGFP-FM4-hGH signal in the perinuclear region.

Model of S-acylation dependent recruitment of NLRP3 to intracellular membranes.

A) Interaction of NLRP3 with intracellular membranes is likely to be driven through a initial transient mode of binding provided by residues in helix115-125 and the polybasic region. Stable enrichment and trapping of NLRP3 at the Golgi requires S-acylation at Cys-130 by ZDHHC enzymes with removal of NLRP3 likely catalysed by proteins from the APT or ABHD family of thioesterases.

Palmitoyltransferase and thioesterase transcript levels in THP-1 cells and HeLa cells.

Data are collated from the human protein atlas (https://www.proteinatlas.org/).