Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo

Abstract

Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Gαo - α-subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Gαo N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

Fig. 1. Key amino acid residues in Gαo N-terminus.

a Sequence of the GFP-fusions of Gαo N-terminus. b–f N2a cells expressing Gαo-GFP (b), Gαo-Nt⁷-GFP (c) or Gαo-Nt³¹-GFP (d) were immunostained against GM130. Color-channels are listed vertically top-to-bottom and selected areas are magnified to the right with the channels displayed vertically in the same order. DAPI stained nuclei in blue. Mean fluorescence intensity ratios of GFP-fusions at the Golgi (e) or PM (f) versus total cell. Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); four independent experiments (Gαo-Nt⁷, n = 56; Gαo-Nt³¹, n = 61; Gαo, n = 58). g, h Subcellular fractionation of constructs described in (b–d). Anti-GFP antibody used to detect Gαo constructs, and anti-GAPDH and anti-flotillin-2 (Flot-2) as cytosolic (Cyto) and membrane (Mem) markers, respectively (g). Distribution of GFP-fusions in cytosolic and membrane fractions (h). Data as mean ± s.e.m.; six independent experiments. i, j Localization of Gαo-G2L-Nt⁷-GFP (i) and Gαo-C3N-Nt⁷-GFP (j) in N2a cells. k, l Fractionation of N2a cells expressing Gαo-Nt⁷-GFP or indicated Nt⁷-mutants (k). Underlined residues depict substitutions in Gαo-Nt⁷. Immunodetection and quantification done as in (g, h). Data represent mean ± s.e.m.; 4–6 independent experiments (l). m, n Localization of Gαo-Nt⁷-GFP under inhibition of N-myristoylation (DDD85646; m) or S-palmitoylation (2-bromopalmitate; 2-BrPal; n). o–q [³H]palmitate radiolabeling of Gαo-Nt⁷-GFP, MGNC-Nt⁷-GFP, and Gαo-C3N-Nt⁷-GFP. Immunoprecipitated (IP) GFP-fusions from control, 2-BrPal and Palmostatin B (PalmB) treated cells were analyzed by autoradiography ([³H]palmitate) and anti-GFP antibody (o). Radioactivity incorporated to constructs normalized to respective controls (p) or Gαo-Nt⁷ (q). Data as mean ± s.e.m.; three independent experiments. r, s Localization of Serine 6 (S6) mutants of Gαo-Nt⁷-GFP under control (r) or DDD85646 (s) treatment. Ser-to-Cys (S6C) mutant showed a higher PM targeting, quantified in (t). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); four independent experiments (Gαo-Nt⁷, n = 56; S6C, n = 63). b–d, i, j, m, n, r, s Scale bars, 10 µm. P values were determined using one-way ANOVA Tukey test for (e, f), two-way ANOVA Tukey test for (h, l), one-sample t-test for (p, q), and two-sided unpaired t-test for (q, t). ns: not significant. Source data are provided as a Source Data file.

Fig. 2. Position of the Cys in Gαo N-terminus.

a–d N2a cells expressing the Nt⁷-GFP mutants MGNC (a) or MGNTC (b) and immunostained against GM130 (a, b). Nuclei in blue stained with DAPI. Color-channels are listed vertically top-to-bottom and selected areas are magnified to the right with the channels displayed vertically in the same order. Underlined letters indicate residues substituted in Gαo-Nt⁷. Mean fluorescence intensity ratios of GFP-fusions at the PM versus total cell (c); four independent experiments (Gαo-Nt⁷, n = 56; MGNC-Nt⁷, n = 50; Gαo, n = 58). Co-localization analysis of the Nt⁷-GFP constructs with GM130 (d); three independent experiments (Gαo-Nt⁷, n = 33; MGNC-Nt⁷, n = 36; MGNTC-Nt⁷, n = 39). e–g N2a cells expressing the MGNC mutant of full-length Gαo (MGNC-Gαo-GFP) and immunostained against GM130 (e). Boxed area is enlarged. Underlined letters indicate residues substituted in Gαo. Relative localization of GFP-fusions at the PM (f); four independent experiments (Gαo, n = 56; MGNC-Gαo, n = 46; MGNC-Nt⁷, n = 50). Co-localization with GM130 (g); three independent experiments (Gαo, n = 47; MGNC-Gαo, n = 38; MGNTC-Nt⁷, n = 36). h Localization in N2a cells of a Nt⁷-GFP construct comprising the consensus sequence of eukaryotic Gα-Nt⁷ with the conserved Cys at position 5 (MGSLCSR). Marked region is magnified to the right. i–k Drosophila S2 cells expressing Gαo-Nt⁷-GFP (i), MGNC-Nt⁷-GFP (j) or MGSLCSR-Nt⁷-GFP (k). Co-expression of GalT-mRFP marked Golgi stacks (i–k). Selected areas are zoomed-in to the right. l–n Live imaging of N2a cells co-expressing Gαo-Nt⁷-GFP (l) or MGNC-Nt⁷-GFP (m) with the Golgi marker MannII-mRFP (bottom right insets). Representative images at the time of Palmostatin B (PalmB) addition (0 min) and after 45 min. Co-localization of Nt⁷-GFP constructs with MannII-mRFP in 5 min intervals and starting at t = 0 (n). Data represent mean ± s.d. (Gαo-Nt⁷ control, n = 20; Gαo-Nt⁷ PalmB, n = 20; MGNC-Nt⁷ control, n = 16; MGNC-Nt⁷ PalmB, n = 20). a, b, e, h, l, m Scale bars, 10 µm; i–k Scale bars, 5 µm. c, d, f, g Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers). P values were determined using one-way ANOVA Tukey test for (c, d, f, g) and one-way ANOVA Šídák test for n. ns: not significant. Source data are provided as a Source Data file.

Fig. 3. Targeting of Nt⁷-GFP.

a, b Reverse dimerization (RD) assay in HeLa expressing Gαo-Nt⁷-FM⁴-GFP (a) or MGNC-Nt⁷-FM⁴-GFP (b). Cytosolic clusters formed by the Nt⁷-FM⁴-GFP constructs dissolve by the addition of D/D solubilizer (D/D-Sol) and show their expected localizations. Underlined letters indicate residues substituted in Gαo-Nt⁷. c–e Live imaging of HeLa cells co-expressing Gαo-Nt⁷-FM⁴-GFP (c) or MGNC-Nt⁷-FM⁴-GFP (d) and the Golgi marker MannII-mRFP (bottom right insets). Representative images of the Nt⁷-FM⁴-GFP constructs at the time of D/D solubilizer addition (0 min) and after 6 min (c, d). Increase upon time of Nt⁷-FM⁴-GFP constructs at the Golgi region (e). Note that Gαo-Nt⁷-FM⁴-GFP quickly targets the Golgi region, while MGNC-Nt⁷-FM⁴-GFP only slightly accumulates at the Golgi. Data represent mean ± s.d. (Gαo-Nt⁷, n = 12; MGNC-Nt⁷, n = 11). f–h RD assay in HeLa cells expressing the secretable control GFP-FM⁴-hGH (f) or MGNC-Nt⁷-FM⁴-GFP (g) performed at 37 °C or at 20 °C to inhibit Golgi transport. Incubation with D/D solubilizer results in the almost complete secretion of GFP-FM⁴-hGH at 37 °C but in Golgi retention at 20 °C (f). PM targeting of MGNC-Nt⁷-FM⁴-GFP seems not affected by the 20 °C temperature block and its presence at the Golgi region (MannII-mRFP) is not higher than at 37 °C (g). Mean fluorescence intensity of MGNC-Nt⁷-FM⁴-GFP, Golgi over total cell (h). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (37 °C, n = 49; 20 °C, n = 40). a–d, f, g Scale bars, 10 µm. P value was determined using a two-sided unpaired t-test for (h). ns: not significant. Source data are provided as a Source Data file.

Fig. 4. Local S-palmitoylation at the ONM—the “SwissKASH” assay.

a Schematic representation of the core components of the SwissKASH assay. b–e Representative images of HeLa cells expressing the constructs mRFP-KASH that targets the ONM (b), mRFP-zDHHC5 (c) or mRFP-zDHHC5-KASH (d, e) with or without the co-expression of SUN2. Note that mRFP-zDHHC5-KASH efficiently targets the ONM in the presence of SUN2 (e). b–e Scale bars, 10 µm.

Fig. 5. The SwissKASH assay for PM and Golgi zDHHCs.

a–e Representative images of the SwissKASH assay in HeLa cells for Gαo-Nt⁷-GFP (a, b) and MGNC-Nt⁷-GFP (c, d) using the control mRFP-KASH construct (a, c) or mRFP-zDHHC11-KASH (b, d). Nuclei stained in blue with DAPI. Color-channels are listed vertically top-to-bottom and selected areas are magnified to the right with the channels displayed also vertically in the same order. Underlined letters indicate residues substituted in Gαo-Nt⁷. Note that the expression of the KASH-construct carrying zDHHC11 results in the strong recruitment of Gαo-Nt⁷-GFP (b) and MGNC-Nt⁷-GFP (d) at the ONM, effect not seen by mRFP-KASH (a, c). Mean fluorescence intensity ratios of GFP-fusions at the ONM versus cytosol (e). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (Gαo-Nt⁷ and mRFP-KASH, n = 44; Gαo-Nt⁷ and zDHHC11, n = 45; MGNTC-Nt⁷ and mRFP-KASH, n = 49; MGNTC-Nt⁷ and zDHHC11, n = 43). f–k The SwissKASH assay using zDHHC3 (f, h) and zDHHC7 (g, i). Gαo-Nt⁷-GFP efficiently targets the ONM by the co-expression of mRFP-zDHHC3-KASH (f) and mRFP-zDHHC7-KASH (g). MGNC-Nt⁷-GFP poorly localizes at the ONM in the presence of mRFP-zDHHC3-KASH (h) and mRFP-zDHHC7-KASH (i). Selected areas are magnified to the right. Mean fluorescence intensity ratios of GFP-fusions at the ONM versus cytosol by zDHHC3 (j) and zDHHC7 (k). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (Gαo-Nt⁷ and mRFP-KASH, n = 44; Gαo-Nt⁷ and zDHHC3, n = 50; Gαo-Nt⁷ and zDHHC7, n = 46; MGNTC-Nt⁷ and mRFP-KASH, n = 49; MGNTC-Nt⁷ and zDHHC3, n = 53; MGNTC-Nt⁷ and zDHHC7, n = 45). l–o ONM recruitment of Gαo-Nt⁷-GFP is not observed in the SwissKASH assay using the inactive DHHS-mutants for zDHHC3 (zDHHS3; l), zDHHC7 (zDHHS7; m), and zDHHC11 (zDHHS11; n). The zDHHS11 mutant shows no ONM recruitment of MGNC-Nt⁷-GFP (o). Boxed areas are zoomed-in to the right. a–d, f–i, l–o Scale bars, 10 µm. P values were determined using one-way ANOVA Šídák test for (e, j, k). ns: not significant. Source data are provided as a Source Data file.

Fig. 6. The zDHHC5-Golga7b complex.

a–d The SwissKASH assay for Gαo-Nt⁷-GFP (a) and MGNC-Nt⁷-GFP (b) using mRFP-zDHHC5-KASH and Golga7b-Flag in HeLa cells. Note that the KASH-fusion of zDHHC5 induces the ONM recruitment of MGNC-Nt⁷-GFP (b) and Golga7b (a, b), but not of Gαo-Nt⁷-GFP (a). ONM localization of MGNTC-Nt⁷-GFP and Golga7b is not induced by the inactive DHHS-mutant of zDHHC5 (zDHHS5; c). Cells were immunostained against Flag-tag and nuclei were stained in blue with DAPI. Marked regions are magnified at the bottom panels. Underlined letters indicate residues substituted in Gαo-Nt⁷. Mean fluorescence intensity ratios of GFP-fusions at the ONM versus cytosol (d). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (Gαo-Nt⁷ and mRFP-KASH, n = 44; Gαo-Nt⁷ and zDHHC5-Golga7b, n = 49; MGNTC-Nt⁷ and mRFP-KASH, n = 49; MGNTC-Nt⁷ and zDHHC5-Golga7b, n = 48). e–i The SwissKASH assay for Gαo-Nt³¹-GFP using the control mRFP-KASH (e), zDHHC3 (f), zDHHC7 (g), and zDHHC11 (h). Color-channels are listed vertically top-to-bottom and selected areas are magnified to the right with the channels displayed also vertically in the same order. Mean fluorescence intensity ratio (ONM versus cytosol) of Gαo-Nt³¹-GFP compared to Gαo-Nt⁷-GFP (i). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (For Gαo-Nt⁷: mRFP-KASH, n = 44; zDHHC3, n = 50; zDHHC7, n = 46; zDHHC11, n = 47. For Gαo-Nt³¹: mRFP-KASH, n = 42; zDHHC3, n = 45; zDHHC7, n = 40; zDHHC11, n = 46). a–c, e–h Scale bars, 10 µm. P values were determined using one-way ANOVA Šídák test for (d, i). ns: not significant. Source data are provided as a Source Data file.

Fig. 7. zDHHC expression levels drive Nt⁷ compartmentalization.

a–d Representative images of N2a cells expressing Gαo-Nt⁷-GFP (a, b) together with HA-zDHHC11 (a) or its inactive DHHS-mutants (HA-DHHS11; b). Golgi was labeled by the co-expression of MannII-BFP and HA-tagged zDHHCs were immunostained against HA (a, b). Color-channels are listed vertically top-to-bottom and selected areas are magnified with the channels displayed horizontally in the same order left-to-right. Mean fluorescence intensity ratio of Gαo-Nt⁷-GFP at the PM versus total cell (c), and co-localization with MannII-BFP (d). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (For c: Control, n = 58; zDHHC11, n = 61; zDHHS11, n = 54. For d: Control, n = 51; zDHHC11, n = 53; zDHHS11, n = 53). e–l N2a cells expressing MGNC-Nt⁷-GFP (e, f, i, j) together with HA-zDHHC3 (e), HA-zDHHS3 (f), HA-zDHHC7 (i), or HA-DHHS7 (j). Underlined residues indicate substitutions in Gαo-Nt⁷. Mean fluorescence intensity ratio of MGNC-Nt⁷-GFP at the PM versus total cell (g, k), and co-localization with MannII-BFP (h, l). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (For g and k: Control, n = 56; zDHHC3, n = 53; zDHHS3, n = 56; zDHHC7, n = 54; zDHHS7, n = 52. For h and l: Control, n = 54; zDHHC3, n = 53; zDHHS3, n = 51; zDHHC7, n = 52; zDHHS7, n = 55). m–o Representative [³H]palmitate metabolic radiolabeling of Gαo-Nt⁷-GFP and MGNC-Nt⁷-GFP upon the co-expression of HA-zDHHC3, HA-zDHHC7, HA-zDHHC11, or HA-zDHHC5 plus Golga7b-Flag. GFP-fusions were immunoprecipitated (IP), and analyzed by autoradiography ([³H]palmitate) and anti-GFP antibody (m). Radioactivity incorporated to Gαo-Nt⁷ (n) and MGNC-Nt⁷ (o) normalized to the respective controls. Data represent mean ± s.e.m.; three independent experiments. a, b, e, f, i, j Scale bars, 10 µm. P values were determined using one-way ANOVA Šídák test for c, d, g, h, k, l and one-sample t-test for (n, o). ns: not significant. Source data are provided as a Source Data file.

Fig. 8. Downregulation of zDHHCs affects Nt⁷ compartmentalization.

a–g Representative images of HeLa cells expressing Gαo-Nt⁷-GFP (a–c) or MGNC-Nt⁷-GFP (d–f). Cells were previously transfected with control siRNA (siControl; a, d), siRNAs against zDHHC3 and 7 (siD3 + siD7; b, e), or against zDHHC5 and 11 (siD5 + siD11; c, f). Cells were immunostained against GM130 to mark the Golgi, and nuclei were stained with DAPI in blue. Color-channels are listed vertically top-to-bottom and selected areas are magnified with the channels displayed also vertically in the same order. Underlined letters indicate residues substituted in Gαo-Nt⁷. Mean fluorescence intensity ratios of GFP-constructs at the Golgi vs total cell (g). Box plots indicate median (middle line), 25th, 75th percentile (box), and lowest, highest value (whiskers); three independent experiments (For Gαo-Nt⁷ (a–c): siControl, n = 57; siD3 + siD7, n = 57; siD5 + siD11, n = 60. For MGNC-Nt⁷ (d–f): siControl, n = 55; siD3 + siD7, n = 58; siD5 + siD11, n = 57). h–i Representative [³H]palmitate metabolic radiolabeling of Gαo-Nt⁷-GFP and MGNC-Nt⁷-GFP from cells pretreated with siControl, siD3 + siD7, and siD5 + siD11. GFP-fusions were immunoprecipitated (IP), and analyzed by autoradiography ([³H]palmitate) and anti-GFP antibody (m). Radioactivity incorporated to Gαo-Nt⁷ and MGNC-Nt⁷ normalized to siControl. Data shown as the mean ± s.e.m.; three independent experiments. a–f Scale bars, 10 µm. P values were determined using one-way ANOVA Šídák test for (g) and one-sample t-test for (i). ns: not significant. Source data are provided as a Source Data file.

Fig. 9. Targeting of full-length Gαo to the ONM.

a–d The SwissKASH assay for full-length Gαo (Gαo-GFP) using the control mRFP-KASH (a), zDHHC3 (b), zDHHC7 (c) and zDHHC11 (d) in HeLa cells. Nuclei stained in blue with DAPI. Color-channels are listed vertically top-to-bottom and selected areas are magnified to the right with the channels displayed also vertically in the same order. Note that the ONM accumulation of Gαo-GFP is induced by all zDHHCs but not the control mRFP-KASH. e, f The full-length Gαo-GFP (e) and its MGNC mutant (MGNC-Gαo-GFP; f) were tested in the SwissKASH system using mRFP-zDHHC5-KASH and Golga7b-Flag. Cells were immunostained against Flag-tag. Marked regions are magnified at the bottom panels. Underlined letters indicate residues substituted in the Nt⁷ region of Gαo. Note that only MGNC-Gαo-GFP (f) and Golga7b (e, f) accumulate at the ONM. g, h The SwissKASH assay applied to Gαo-GFP (g) and its GTPase-inactive mutant Q205L (h) together with mRFP-Gβ1γ3 (g) or His₆-tagged RGS19 (His-RS19; h), and using BFP-zDHHC11-KASH (not displayed). Cells were immunostained against the His₆-tag (h). Selected areas are magnified at the bottom panels. a–h Scale bars, 10 µm.

Fig. 10. Model of compartmentalization of PMPs via local S-palmitoylation.

Model of how some peripheral membrane proteins might achieve specific subcellular compartmentalization by the interplay of the substrate selectivity of different zDHHCs. The model uses Gαo-Nt⁷-GFP (green circles) and MGNC-Nt⁷-GFP (cyan circles) as examples.