S-Palmitoylation Sorts Membrane Cargo for Anterograde Transport in the Golgi

Abstract

While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.

Keywords: DHHC; Golgi; S-palmitoylation; cargo sorting; trafficking.

Figure 1.. Anterograde transported cargo is S-palmitoylated in the cis-Golgi, and Golgi-localized DHHCs are concentrated in the cis.

A HeLa cells were metabolically labeled with 50 μM alkyne-palmitate for 10 min, fixed, permeabilized, clicked to azideAF647, immunostained for endogenous Golgi markers (cis-Golgi: GPP130, trans-Golgi: p230), and subjected to structured illumination super-resolution microscopy (3D-SIM, max. intensity Z-projections; scale bars: 2 μm). B Labeling of the Golgi with alkynepalmitate requires CoA-activation, DHHC enzyme function, and the presence of biosynthetic cargo. Pretreatment of cells with Triacsin C (TriaC, 100 μM, inhibitor of the long chain fatty acyl-CoA synthetase), 2-bromopalmitate (2-BP, 100 μM, competitive DHHC inhibitor), or cycloheximide (CHX, 100 μg/ml, inhibitor of protein biosynthesis) for 45 min prior to a 10 min pulse of alkyne-palmitate prevents Golgi labeling. The average integrated intensity of alkyne-palmitate signal at the Golgi in presence of inhibitors was quantified and compared to mock-treated controls (n>60 cells/timepoint and condition, mean and SEM, two-tailed unpaired t-tests (***: p<0.001)). C 3D-SIM of alkynepalmitate with cis- and trans-Golgi markers for a 5 or 10 min pulse (50 μM), followed by a chase with natural palmitate at 10x molar excess after 10 min. Co-localization (Pearson´s R) was determined and subjected to two-tailed, unpaired t-tests (***: p<0.001; n>10 cells/timepoint, mean and SEM). D Hela cells were constantly labeled with 50 μM alkyne-palmitate, or pulsed for 10 min followed by a chase with natural palmitate at 10x molar excess (500 μM). After click chemistry, the integrated fluorescence intensity of the Golgi area was quantified (n>20 cells/timepoint and condition, mean and SEM). E Co-localization analysis of human DHHC 3 and 7 (3DSIM). SNAP-DHHC isoforms were transfected for 24 h. After fixation, endogenous Golgi markers were immunolabeled (cis-Golgi: GPP130, trans-Golgi: p230), and SIM images of the Golgi area were obtained (max. intensity Z-projections are given, scale bars: 2 μm). F Localization of human DHHC candidates within the Golgi. SNAP-DHHC fusions of 9 Golgi candidates were scored for co-localization as described in E, and subjected to two-tailed, unpaired t-tests (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; n>20 cells/timepoint, mean and SEM). G Schematic of the localization of human DHHC palmitoyltransferases within the Golgi. See also Fig. S1.

Figure 2.. DHHCs 3 and 7 account for the majority of S-palmitoylated cargo proteins at the Golgi and catalyze their transport to the plasma membrane.

A Human SNAP-tagged DHHC candidates were transiently transfected into HeLa cells and metabolically labeled with azide-palmitate for 20 minutes. The ratio of the integrated intensity of palmitate and the respective DHHC was quantified (n>10, mean and SEM). B HeLa cells overexpressing SNAP-DHHC3 were pretreated with Triacsin C (TriaC, 100 μM), 2-bromopalmitate (2-BP, 100 μM), or cycloheximide (CHX, 100 μg/ml) prior to a 10 min pulse with alkyne-palmitate. Confocal Z-stacks were obtained, and the ratio of palmitate to DHHC fluorescence was quantified as in A (n>30 cells per timepoint and condition, mean and SEM). C Cells transfected with DHHC isoforms 3 and 7, or their enzymatically inactive (DHHS) variants were labeled with alkyne-palmitate for 20 min. To estimate the fraction of thioester-bound palmitate in the Golgi area, the samples were incubated with 1.5M neutral hydroxylamine for 2h followed by an extensive washout. The ratio of palmitate and DHHC fluorescence was quantified, normalized to the respective wt construct, and subjected to two-tailed unpaired t-tests (***: p<0.001, n>50 cells per condition, mean and SEM). D Overexpressed DHHCs 3 and 7 catalyze S-palmitoylation of numerous protein substrates. Cells expressing SNAP-DHHC3, 7, or mock-transfected cells were labeled for 20 min with alkyne-palmitate, clicked to azideAF647, and subjected to in-gel fluorescence analysis. E-F Cells were transfected with DHHC isoforms 3 (E), 7 (F), or their inactive (DHHS) variants, metabolically labeled with alkyne-palmitate for 10 min, and subjected to a chase with natural palmitate at 10x molar excess for the indicated time. After fixation, permeabilization, and labeling, the samples were subjected to TIRF microscopy (n>20 cells per construct, timepoint, and condition, mean and SEM). See also Fig. S2–4.

Figure 3.. Palmitoylation of membrane proteins accelerates their rate of intra-Golgi protein transport.

A HeLa cells transfected with wt VSVG-GFP or its non-palmitoylated variant VSV G-C490-GFP were shifted from non-permissive (40.5°C) to permissive temperature (32°C) to generate an anterograde wave of cargo. A Upper left panel: Surface arrival of S-palmitoylated VSV G is accelerated. Cells transfected with VSV G or C490A were released from the ER, and subjected to surface biotinylation with sulfoNHS-biotin at the indicated timepoint. A ratio of surface to total was quantified for n=3 independent experiments (mean and SEM). A Upper right panel: S-palmitoylation of VSV G does not impact the rate of entry into the Golgi. Constructs were released from the ER, and the rate of acquisition of EndoH-resistant N-glycans was monitored via Western blot. Quantification of n=4 independent experiments (mean and SEM). A Lower left panel: Surface arrival does not differ after release from the trans Golgi/TGN. Cargo was arrested in the trans-Golgi via a 20°C bl ock for 2h prior to releasing the temperature block for the indicated times. Quantification of n=3 independent experiments (mean and SEM). A Lower right panel: Pull-down experiments employing the Galactose-specific lectin Jacalin. Quantification of n=3 independent experiments, normalized to wt G peak at 10 min (mean and SEM). B HeLa cells transfected with transferrin receptor (TfR-FM4-HALO) or its non-palmitoylated variant (TfR-C62AC67AFM4-HALO) were released from the ER using a cell-permeable small molecule solubilizer (D/D) to generate an anterograde wave of cargo. B Upper left panel: the rate of surface arrival was quantified employing live-cell TIRF imaging. The integrated intensity per cell over time from n=4 independent experiments per construct and condition was quantified, and normalized to initial fluorescence intensity (mean and SEM). B Upper right panel: Co-localization with a cis Golgi marker (HPL) after release from the ER for the indicated times. Quantification of n>20 cells per timepoint and construct (mean and SEM). B Lower left panel: Surface arrival of TfR constructs does not differ when released from trans Golgi/TGN. Live TIRF imaging of cells transfected with TfR constructs was performed after a 20°C bloc k for 2h in presence of D/D solubilizer. Quantification of n>5 cells per construct (mean and SEM). B Lower right panel: Acquisition of Galactose in the trans Golgi is accelerated for palmitoylated TfR. Acquisition of Galactose in the trans Golgi was monitored as for VSV G in A by employing Jacalin. Quantification of n=3 independent experiments (mean and SEM). See also Fig. S5.

Figure 4.. Modulation of DHHC levels and their activity impacts transport of both membrane and soluble proteins.

A-B Modulation of VSV G surface arrival by overexpression of SNAP-DHHC3, the competitive DHHC inhibitor 2-bromopalmitate (2BP), or the de-palmitoylation inhibitor palmostatin B (PB). HeLa cells were transfected with VSV G (A) or C490A (B) for 24 h and incubated at 40.5°C. If indicated, e xpression of DHHC3 was induced in the stably transfected cells for 5h. Un-induced, VSV Gtransfected parallel setups were incubated for 1 h with 2-BP (100 μM) or PB (25 μM). VSVG was released from the ER for 30 min prior to biotinylation of surface-exposed proteins. Quantification of n=3 independent experiments, mean and SEM. The results of two-tailed, unpaired t-tests are given (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001). C Overexpression of DHHC3 modulates the secretion of soluble cargo. The model protein hGh-FM4-GFP was transfected into dox-SNAP-DHHC3 cells. Expression was induced for 5h, prior to releasing hGh from the ER for the indicated times using D/D solubilizer. Cell contents were compared to hGh in the medium after chloroformmethanol precipitation (quantification of n=3 independent experiments, mean and SEM). Upon entry into the Golgi, S-palmitoylated TfR segregates with palmitate into areas enriched with coatomer. D-E 3D-SIM of TfR-FM4-SNAP (D) or TfRC62AC67AFM4-SNAP (E) 15 min post release from the ER with a simultaneous metabolic labeling using alkyne-palmitate. After fixation, S-palmitoylated proteins were subjected to CuAAC to azide-AF647, and SNAP-tagged TfR constructs (AF488) and endogenous coatomer (CM1, AF568) were immunolabeled. F Quantification of co-localization (Pearson’s R) of TfR constructs with palmitate in the cis Golgi (15 min post release from the ER, n=30 cells/construct, SIM max. intensity Z-projections, normalized to wt TfR). G Quantification of the ratio (integrated intensity) of palmitate and coatomer per TfR construct and for the Golgi area as in F. H Zoom into Golgi area indicating enlarged cisternal rims (15 min post release of TfR from the ER). White arrowheads: putative carriers positive for TfR, palmitate, and encompassed with coatomer (lower arrow), TfRenriched rim area (upper arrow). Scale bar: 1 μm.

Figure 5.. S-Palmitoylated membrane cargo concentrates in highly curved regions at the cisternal rims of the Golgi.

A STED nanoscopy of S-palmitoylated proteins at the Golgi. doxSNAP-DHHC3 cells were induced for 5 h and subsequently metabolically labeled with alkyne-palmitate for 20 min, followed by a 10 min chase in delipidated medium. After fixation and permeablilization, palmitoylated proteins were detected with azide-ATTO594, and coatomer or GM130 were immunolabeled with ATTO647N. Left: overview of Golgi area labeled for STED microscopy. Upper right: zoom into individual cisternal elements (dashes boxes i & ii). Lower right panel: line profiles obtained from the magnified images (dashed lines, arrowheads highlight the position of distinct COPIclusters). B Quantification of STED images (cumulative frequency distribution of the proximity of pixels) obtained from Golgi areas (dashed line: 100nm; n=10 Golgi areas per pair, mean and SD). C Electron tomography of S-palmitoylated proteins at the Golgi. Expression of DHHC3 and labeling of the Golgi with alkyne-palmitate was performed as in A, and followed by fixation and permeabilization of the cells. After click chemistry to azide-biotin, S-palmitoylated proteins were detected using streptavidin-fluoronanogold and subjected to double tilt electron tomography. 3D-modelling of the tomogram data was performed (Golgi membranes) and the loci of 3575 gold-labeled palmitoylated proteins in the tomogram were annotated. Magnification of a cisternal rim area of palmitate-labeled Golgi membranes (3D tomogram model). Yellow: gold clusters (=palmitoylated proteins), blue: rendered Golgi membranes, green: vesicular/tubular structures; scale bar: 50 nm. Full model: Fig. S6E. D Local curvature was assessed at each point along the annotated membrane curves, and the cumulative frequency of total Golgi membrane curvature (dashed line) is compared to the underlying curvature observed in loci positive for S-palmitoylated proteins (solid line). See also Fig. S6.

Figure 6.. Curvature preference may be an intrinsic property of palmitoylated anterograde cargo.

A Schematic representation of a minimal construct of the transferrin receptor, TfRmini. Red: Cys residues 62 and 67, grey: transmembrane domain, green: linker and FLAG epitope. B Micromanipulation of giant unilamellar vesicles (GUVs). GUVs containing biotinylated and fluorescently-labeled lipids were formed, and recombinant proteins were reconstituted into GUVs. Next, two micropipettes were employed, one containing a streptavidin bead, one to exert suction on the GUV while pulling a tube out of the membrane due to the biotin-streptavidin interaction (upper panel). The ratio of the fluorescence of the recombinant protein in the tubule versus the GUV donor membrane divided by the intensity of membrane lipids in tubule versus the donor membrane was calculated (representative individual channels for S-palmitoylated or non-palmitoylated TfRmini: middle panels). The model tubes exhibit identical sum-of-lipid pixel intensities in the lipid channel and thus allow the direct comparison of sum of protein pixel intensities (lower panels; tube/GUV enrichment: +50% (S-palmitoylated TfRmini); −20% (de-palmitoylated TfRmini)). C S-palmitoylated TfRmini partitions into tubules with high curvature. TfRmini-containing GUVs were micromanipulated to generate different classes of tube diameters, and the relative excess of recombinant protein in the tube was plotted against increasing tube diameter bins for palmitoylated and de-palmitoylated (hydroxylamine-cleaved) TfRmini. See also Fig. S7.

Figure 7.. S-palmitoylation acts as a sorting signal for anterograde membrane cargo at the cis-Golgi.

Upon interaction with DHHCs in the cis-Golgi, anterograde membrane cargo is rapidly S-palmitoylated. This leads to an increased affinity for the highly positively curved cisternal rim, from which budding occurs, and an extraction of cargo from stacked (flat) Golgi membranes. Partitioning into in curved regions at the rims of the Golgi cisternae increases the incorporation of S-palmitoylated proteins into coatomer-positive carriers, resulting in an acceleration of anterograde transport across the Golgi.