substrate specificities are far from being well understood, although substantial advance has been made lately 12 .
S-palmitoylation of peripheral membrane proteins (PMPs), including Gα subunits, was shown to occur exclusively at the Golgi, where palmitoylated proteins are subsequently transported to the PM 13 . In recent years, however, cumulative data has emerged indicating that some PMPs might undergo local S-palmitoylation on their target compartments, namely the PM or ER 14, 15 . These opposing views appear difficult to reconcile primarily due to the lack of experimental methods allowing visualization of S-palmitoylation in intact cells.
Here, we defined the critical parameters that govern Gαo N-myristolation, S-palmitoylation and subcellular compartmentalization. By engineering a system that allows the ectopic localization of zDHHCs to the outer nuclear membrane, we showed an intriguing substrate specificity of several zDHHCs towards closely related substrates. Moreover, our data indicate that the steady-state localization of Gαo at the PM and Golgi apparatus is the outcome of local S-palmitoylation events. These findings contrast the traditional views that i) Spalmitoylation of PMPs occurs exclusively at the Golgi and ii) serves to drive subsequent PM-directed delivery of such proteins. The unexpected selectivity among different PATs and their substrates we uncover to drive the intracellular localization of PMPs emerges as an attractive target for drug discovery.
For its membrane association, the N-terminus of Gαo is lipid modified by N-myristoylation at Gly2 and S-palmitoylation at Cys3 7 . A recent structural analysis identified the recognition sequence of N-Myristoyltransferases (NMTs) as an N-terminal hexapeptide, excluding Met1 16 . This suggest that a minimal membrane-binding information might reside within the first seven residues of Gαo. Thus far, three crystal structures of heterotrimeric Go have been solved [17] [18] [19] , showing a prominent α-helix in the Gαo N-terminus that extends toward the Gβγ heterodimer. An overlay of the N-terminal region of these three structures revealed a similar ending of the α-helixes, whereas their beginning falls on different residues at positions between 6 to 10 ( Supplementary Fig. 1a-c) . A similar overlay of the N-terminal region of seven solved structures of Gαi1 -a close Gαo homologue -showed a much more consistent beginning for the α-helix, which starts at residues 7 or 8 ( Supplementary Fig. 1d-f ). To study if these regions might have specific roles in Gαo membrane association, we generated the following GFP-fusion constructs (Fig. 1a) : one including Gαo N-terminal heptapeptide (Gαo-Nt 7 -GFP), another with the first 31 residues covering the α-helix (Gαo-Nt 31 -GFP), and the third containing only the α-helix (Gαo-Nt 8-31 -GFP). These constructs were expressed in the mouse neuroblastoma Neuro-2a cells (N2a) and their localization at the plasma membrane (PM) and Golgi apparatus were compared with the full-length Gαo-GFP 6 . Surprisingly, Gαo-Nt 7 -GFP localized predominantly at the Golgi, whereas Gαo-Nt 31 -GFP displayed a more homogenous PM and Golgi distribution similar to Gαo-GFP (Fig. 1b) . Quantification of average fluorescence intensities at these compartments confirmed a much higher Golgi and a lower PM localization of Gαo-Nt 7 -GFP compared to Gαo-Nt 31 -GFP and Gαo-GFP ( Fig.   1c ,d). Then, we performed a crude subcellular fractionation of N2a cells expressing the constructs and showed that Gαo-Nt 7 -GFP, Gαo-Nt 31 -GFP and Gαo-GFP were similar in the way they partitioned between the cytosolic and membrane fractions (Fig. 1e,f) . As opposed to these constructs showing prominent membrane localizations, Gαo-Nt 8-31 -GFP was spread over the cytosol and nucleus ( Supplementary Fig. 1g ), indicating that the N-terminal α-helix alone is not sufficient for membrane association.
In the heterotrimeric G protein complex, the N-terminal α-helix of Gα is one of the regions binding to Gβ 20, 21 . Since Gβ tightly interacts with Gγ, which in turn associates to membranes via its C-terminal prenylation 22 , we tested if the poor PM localization of Gαo-Nt 7 -GFP relates to a lack of Gβγ interaction. For this aim, we co-expressed the Gαo constructs together with mRFP-Gβ1 and mRFP-Gγ3 in N2a cells, and immunoprecipitated the GFP-fusions. Fulllength Gαo-GFP strongly pulled down Gβ1 and Gγ3, whereas Gαo-Nt 7 -GFP and Gαo-Nt 31 -GFP showed a very faint co-precipitation of Gβγ with no apparent difference between them ( Supplementary Fig. 1h) . Thus, the preferential localization of Gαo-Nt 7 -GFP to the Golgi, as opposed to the dual localization of Gαo-Nt 31 -GFP to Golgi and PM, is independent from Gβγ.
Together, these results indicate that Nt 7 is sufficient for Gαo overall membrane binding, but it seems to drive the Golgi rather than the PM localization.
Key residues in Gαo-Nt 7 We next aimed to decode the intracellular localization of Gαo-Nt 7 by a systematical point mutation analysis of its key residues. Amino acid substitutions were designed using the GPS-Lipid prediction tool 23 . For instance, we introduced the Gly-to-Leu mutation at position 2 of Gαo-Nt 7 -GFP as GPS-Lipid predicted very high likelihood for S-palmitoylation at Cys3 in the mutant (not shown). However, the Gly2 mutation led to a complete loss of membrane association as Gαo-G2L-Nt 7 -GFP evenly spread over the cytosol and nucleus in N2a cells ( Fig. 1g ). This result reinforces the notion that N-myristoylation is a prerequisite for Spalmitoylation in Gαo 7 . Consequently, Gαo-G2L-Nt 7 -GFP appeared almost exclusively at the cytosolic fraction (Fig. 1h,i) . We then introduced the Cys-to-Asn mutation at position 3 as GPS-Lipid predicted a high score for N-myristoylation at Gly2 in the mutant (not shown). The resulting Gαo-C3N-Nt 7 -GFP was predominantly excluded from the nucleus and showed an ER-like distribution with certain accumulation at the Golgi, which might be accounted for by the high membrane density of the cisternae stack (Fig. 1g) . Nevertheless, Gαo-C3N-Nt 7 -GFP appeared mostly at the cytosolic fraction (Fig. 1h,i) , confirming that N-myristoylation confers only weak membrane binding properties 8 . Importantly, the localization of Gαo-Nt 7 mutants impaired in N-myristoylation and S-palmitoylation was emulated by the treatment with the specific inhibitors DDD85646 24 and 2-bromopalmitate (2-BrPal) 25 , respectively (Fig. 1j) .
The most recent substrate recognition motif for NMTs was described as
[KR] 7 , with ^ denoting exclusion of residues, and X -any amino acid 16 The authors did not exclude the possibility that peptides lacking a positive charge at position 7 might also be accepted by NMTs. Thus, N-myristoylation of Gαo -and all human N-myristoylated Gα subunits -is not in conflict with the lack of a Lys/Arg residue at this position (Fig. 1a) . At position 6, a Ser residue is found in Gαo and all its homologues, and Ser is by far the most frequent residue at this position among NMT substrates 16 . Thus, we first introduced point mutations at Ser6 in Gαo-Nt 7 based on the substrate recognition motif described by Castrec et al. and on a previous study indicating that Arg, Asn, Phe, and
Val can occupy this position as well 26 . Substitution of Ser6 by Ala, Cys, Gly, or Thr displayed efficient membrane binding in N2a cells (Fig. 1k) . Interestingly, the S6C mutant showed a much higher PM localization compared to Gαo-Nt 7 -GFP (Fig. 1k,l) , suggesting that Cys3 and Cys6 both undergo S-palmitoylation and that PM targeting might be enhanced by the dual Spalmitoylation. N-myristoylation of these mutants was indirectly verified with the inhibitor DDD85646 (Fig. 1k) . Conversely, we observed a largely cytosolic and nuclear localization for the constructs in which the Ser6 was substituted by Arg, Asn, Phe and Val ( Supplementary   Fig. 1i) , suggesting that these residues in position 6 make the N-terminal peptide of Gαo a poor substrate for MNTs.
Intrigued by the S6C mutant, we questioned if the position of the Cys residue within Gαo-Nt 7 might play any role in its subcellular localization. Thus, we moved the Cys3 to positions 4 (MGNC-Nt 7 ) and 5 (MGNTC-Nt 7 ), and expressed the GFP constructs in N2a cells. An Asp residue was placed at position 3 following the GPS-Lipid prediction tool (not sown).
Unexpectedly, MGNC-Nt 7 -GFP displayed a completely different localization than its parental Gαo-Nt 7 (Figs. 1b, 2a). It exhibited PM localization that was not only higher than that of Gαo-Nt 7 -GFP, but also of the full-length Gαo-GFP (Figs. 1b, 2b). Moreover, the perinuclear structures labelled by MGNC-Nt 7 -GFP showed a much lower co-localization with the Golgi marker GM130 compared to Gαo-Nt 7 -GFP (Pearson's correlation; Figs. 1b, 2a,c). Instead, the perinuclear structures containing MGNC-Nt 7 -GFP co-localized better with Lactadherin-C2 (mRFP-Lact-C2; Supplementary Fig. 1j ,k), a biosensor for phosphatidylserine 27 . These results imply that MGNC-Nt 7 -GFP preferentially associates with the PM-derived endocytic rather than the Golgi-derived secretory pathway. The distinct localization of MGNC-Nt 7 -GFP was not the result of a major change in the overall membrane binding, as its presence in the membrane fraction was not significantly different from that of Gαo-Nt 7 -GFP (Fig. 1h ,i). S-Palmitoylation of Cys4 in MGNC-Nt 7 -GFP was confirmed by the incubation with the inhibitor 2-BrPal (Fig. 2d) . On the other hand, the Cys5 construct MGNTC-Nt 7 -GFP localized at the Golgi and cytosol (Fig. 2a ,c) but appeared mostly in the cytosolic fraction upon cell fractionation (Fig. 1h,i) . Thus, it seems that the Cys5 is not a good substrate for palmitoyl acyltransferases (PATs), as MGNTC-Nt 7 -GFP closely resembles the C3N mutant ( Fig. 1g-i) .
Most intriguingly, the subcellular localization of MGNC-Nt 7 in N2a cells was fully recapitulated in the full-length Gαo when the same mutation was introduced. Specifically, MGNC-Gαo-GFP showed a higher PM targeting (Figs. 1b, 2e,f) and a much lower co-localization with the Golgi marker GM130 (Figs. 1b, 2e,g) than Gαo-GFP.
Altogether, these data uncover a remarkable characteristic of the N-terminal localization code of Gαo: minor changes in its amino acid sequence can drastically modify the subcellular localization of the protein. Due to its homology with all dual lipidated Gα subunits, and to other similarly modified PMPs, we reasoned that this principle might have a much broader relevance in biology.
Next, we wanted to determine how much diversity is present in Nt 7 among eukaryotic Gα subunits. We searched for Gα sequences containing an N-terminal heptapeptide with the signature M 1 GXXX[ACGST]X 7 , with at least one Cys at positions 3-5 or 7. Of note, we did not find any Gα subunit orthologue in the clades Ancyromonadida, Apusozoa, Breviatea, Cryptophyceae, CRuMs, Glaucocystophyceae, Malawimonadidae, and Rhodophyta (red algae). We found, however, Gα subunits in abundance in Amoebozoa, Discoba, Haptista, Metamonada, Opisthokonta, Sar, and Viridiplantae. Overall, we identified more than 800 sequences containing the desired Nt 7 signature (Supplementary Table 1 We asked whether consensus sequences might emerge from the Gα subunits among each category. As available databases are biased towards certain clades, we first selected unique hexapeptides with the signature M 1 GXXX[ACGST] 6 . When an identical combination within positions 3-5 was found, we selected the sequence with the most frequent residue at position 7 for this analysis. Thus, we identified 88 unique combinations for Cys3, 75 for Cys4, and 13 for Cys5 (Supplementary Table 2 ). As seen in the corresponding Logo representations, there is no obvious Gα-Nt 7 consensus for the Cys3 and Cys4 categories (Fig. 2h,i) , whereas the sequence MGSLCSR emerges for Cys5 (Fig. 2j) . Plotting the amino acids by their polarity groups showed a common characteristic among categories: positively and even more so negatively charged amino acids are strongly underrepresented at positions 3-5 compared to a random distribution, or even completely absent as in the Cys5 category ( Supplementary   Fig. 2a-d) . One can speculate that charged amino acids might interfere with at least one of the lipid modifications of Gα-Nt 7 . The most recent recognition motif for NMTs (see above)
denotes that positive and negative residues are not favored at certain positions 16 . Accordingly, N-myristoylation of Gαo-Nt 7 -GFP appeared strongly impaired in N2a cells when different combinations of Arg, Asp, Glu, or Lys were introduced at positions 4 and 5 ( Supplementary Fig. 2e) . A Gαo-Nt 7 -GFP mutant with a His occupying position 5 showed a near normal membrane association regardless of the presence of a positive or negative residue at position 4 ( Supplementary Fig. 2f) . Thus, N-myristoylation of Gα-Nt 7 of the Cys3 category appears optimal when Arg, Asp, Glu, or Lys residues are excluded from position 5, in total agreement with the substrate recognition motif described by Castrec et al.
As a consensus emerged for the Cys5 category (Fig. 2j) , we expressed a GFP-fusion of this sequence in N2a cells. Somehow predictably, MGSLCSR-Nt 7 -GFP showed a much better membrane binging than our designed MGNTC-Nt 7 -GFP, and localized predominantly at the Golgi (Fig. 2a,k) . When MGSLCSR was introduced into the full-length Gαo, however, the membrane association of this mutant was not very efficient ( Fig. 2l) , probably related to the fact that Gα subunits of the Cys5 category are virtually absent in Metazoa (Supplementary Table 1 ).
Then, we expressed our constructs Gαo-Nt 7 -GFP, MGNTC-Nt 7 -GFP and the consensus MGSLCSR-Nt 7 -GFP in the Drosophila Schneider-2 cell line. Remarkably, all constructs retained the same localization patterns as seen in N2a cells: Gαo-Nt 7 and MGSLCSR-Nt 7 targeted mainly Golgi stacks labelled by GalT-mRFP, while MGNTC-Nt 7 associated largely with the PM (Fig. 2m) . Similar localizations were also seen in HeLa cells ( Supplementary Fig.   2g ). These data point to highly conserved rules for N-myristoylation and S-palmitoylation as well as for substrate compartmentalization.
Our next goal was to understand the molecular mechanism behind the contrasting subcellular localization of two rather similar sequences: Gαo-Nt 7 and MGNC-Nt 7 . Nmyristoylation generally occurs during protein synthesis and confers a weak membrane binding to the modified protein 25 . N-myristoylation is essential for the subsequent Spalmitoylation to occur 7 . Our own data showed that myristoylated, but not palmitoylated Gαo-Nt 7 and MGNC-Nt 7 are indistinguishable in their localization and only prevented from free cytosolic and nuclear diffusion (Figs. 1j, 2d). Therefore, we speculated that S-palmitoylation is key for subcellular compartmentalization, by itself or in combination with the myristoyl group and/or neighboring amino acids. It has been shown that S-palmitoylation of PMPsincluding Gα subunits -occurs exclusively at the Golgi apparatus, the event followed by the transport of PMPs to the PM via the secretory pathway 13 . Thus, one plausible explanation for the distinct steady-state localizations of Gαo-Nt 7 and MGNC-Nt 7 might involve a differential Golgi-to-PM transport of the constructs.
To test this idea, our first approach was to visualize how these constructs are trafficked in the cell. We adapted to our constructs the reverse dimerization (RD) assay, originally designed for the synchronized trafficking of secretory and integral membrane proteins 28 Altogether, these results imply that the characteristic steady-state localizations of Gαo-Nt 7 and MGNC-Nt 7 are not related to the Golgi-to-PM trafficking. Instead, each of the constructs goes directly to its primary destination: Golgi or PM.
Thus far, our data indicate that S-palmitoylation might contribute to the compartmentalization of Gαo-Nt 7 and MGNC-Nt 7 , and that intracellular trafficking is not a major player in their distinctive localizations. We then envisioned two further scenarios that could explain the differential Nt 7 compartmentalization: (i) PATs have no specificity for substrate recognition, and the substrates concentrate at different compartments due to specific interactions, and (ii) PATs discriminate substrates, which in turn accumulate at the compartment where Spalmitoylation takes place. The first scenario involves a certain degree of promiscuity among PATs 12 , and implies that lipidations and a few surrounding amino acids confer specific binding properties e.g. to lipids, which are known to differentially accumulate all over the endomembrane system 31 . Additionally, fatty acids other than palmitate can also be attached to the available Cys and might confer different binding specificities to the S-acylated protein 32 . The second scenario requires that PATs discriminate between analogous substrates as small as Nt 7 and that a given substrate accumulates at the compartment where its specific PAT is localized.
To test the first scenario, we used commercially available membrane strips spotted with fifteen different lipids found in cellular membranes and performed a protein-lipid overlay assay 33 . As control, we employed a GFP-fusion of the pleckstrin homology (PH) domain of FAPP1 (FAPP1-PH-GFP), that specifically binds to phosphatidylinositol 4-phosphate (PI4P) 34 . N2a cells were transfected with Gαo-Nt 7 -GFP, MGNC-Nt 7 -GFP or FAPP1-PH-GFP, and then membrane strips were incubated with cleared cell extracts. As expected, FAPP1-PH-GFP showed a strong binding to the spot containing PI4P (Supplementary Fig. 3a) . On the other hand, no apparent difference was found for Gαo-Nt 7 -GFP and MGNC-Nt 7 -GFP that mainly bound to phosphatidylserine, cardiolipin, and phosphoinositides, particularly to PI4,5P2 ( Supplementary Fig. 3a) . A similar lipid-binding pattern was also observed by the larger construct Gαo-Nt 31 -GFP ( Supplementary Fig. 3a) . Thus, the compartmentalization of Gαo-Nt 7 and MGNC-Nt 7 is unlikely to emerge from different lipid-binding affinities, arguing against the first scenario.
Next, we studied the second scenario, which implies that local S-palmitoylation drives substrate compartmentalization. In mammals, intracellular S-palmitoylation is mediated by 23-24 zDHHC proteins with PAT activity 11 . Most of the zDHHCs have four transmembrane domains (TMDs), whereas only two and three members present five and six TMDs, respectively 12 . The majority of zDHHCs localize at the Golgi and the remaining are distributed among the ER, PM and endosomes 35, 36 . In order to confirm their subcellular localization, we expressed a collection of twenty-three HA-tagged mouse zDHHCs in N2a cells 37 . As expected, immunostainings revealed that a large number of zDHHCs localized predominantly at the Golgi: zDHHC3, 7, 9, 12, 13, 15-17, 21, 23, and 25 ( Supplementary Fig. 3b ). Six showed a clear PM localization but were also found on endosomes: zDHHC2, 5, 11, 14, 18, and to a lesser extent zDHHC8. The rest of the PATs mostly showed an ER localization, while zDHHC4 associated almost exclusively with the nuclear envelope ( Supplementary Fig.   3b ), a pattern not described in previous studies 35, 36 .
Beside from a unique antibody that specifically recognizes palmitoylated PSD-95 38 , experimental approaches to study zDHHC-substrate pair relations are based exclusively on disruptive biochemical and proteomic techniques 39, 40 . To determine if Gαo-Nt 7 and MGNC-Nt 7 are differently lipidated at the PM vs. Golgi, we aimed at developing a method to visualize local S-palmitoylation in intact cells. Inspired by the nuclear envelope localization of zDHHC4, we engineered zDHHCs that ectopically target the outer nuclear membrane (ONM) to detect mislocalized substrates. We took advantage of some components of the wellstudied LINC (linker of nucleoskeleton and cytoskeleton) complexes. LINC complexes are built by proteins of the KASH (Klarsicht, ANC-1, and Syne Homology) family that are embedded in the ONM and interact within the perinuclear space with proteins of the SUN (Sad1 and UNC-84) family, which in turn are inserted in the inner nuclear membrane 41 . As the C-terminus of all zDHHCs except zDHHC24 faces the cytosol, we hypothesized that addition of a C-terminal KASH-domain might induce their targeting to the ONM (Fig. 4a) . To test this hypothesis, we first generated a zDHHC5 construct carrying an N-terminal mRFP for visualization and the KASH-domain of syne-1/nesprin-1 at its C-terminus (mRFP-zDHHC5-KASH; Fig. 4a ). In HeLa cells, the KASH-domain alone (mRFP-KASH) is efficiently targeted to the ONM (Fig. 4b) , and mRFP-zDHHC5 localized at the PM and endosomes as expected ( Fig. 4b and Supplementary Fig. 3b ). The addition of the KASH domain strongly impaired PM localization of zDHHC5, but a robust ONM targeting was only achieved by the co-expression of the KASH-interacting protein SUN2 (Fig. 4b) .
Next, we analyzed if known zDHHC-substrate pair relations can be recapitulated in HeLa cells and chose the PMPs SNAP23, caveolin-1 and flotillin-2. The core of SNAP23 contains 6
Cys residues that are plamitoylated by zDHHC13 and 17 42 . zDHHC7 appears as the main PAT for caveolin-1, which is plamitoylated in 3 Cys residues at its C-terminus 43 . Flotillin-2 is myristoylated at Gly2 and palmitoylated at Cys4, 19 and 20 by zDHHC5 44 . Remarkably, the GFP-fusions of SNAP23 ( Supplementary Fig. 4a ), caveolin-1 (Supplementary Fig. 4b ) and
flotillin-2 ( Supplementary Fig. 4c) were efficiently recruited to the ONM by the corresponding zDHHC-KASH constructs. Together, we successfully developed a system in intact cells for the visualization of local S-palmitoylation at the ONM (henceforth SpONM).
Once the SpONM system was established, we first tested if PM-associated zDHHCs showed differential substrate specificities for Gαo-Nt 7 and MGNC-Nt 7 . In addition to zDHHC5, the Cterminal KASH-domain effectively targeted zDHHC2, 8, 11, 14 and 18 to the ONM (Fig. 4c,d and Supplementary Fig. 4d) . Remarkably, Gαo-Nt 7 -GFP and MGNC-Nt 7 -GFP were efficiently recruited to the ONM by the co-expression of mRFP-zDHHC11-KASH but not by the control mRFP-KASH (Fig. 4c,d) . To quantify these effects, we measured the mean GFPfluorescence intensity at the ONM and nearby cytosol, and used their ratio to determine the relative ONM content of the Nt 7 constructs. Quantification revealed a similar ~2.5-fold accumulation of Gαo-Nt 7 -GFP and MGNC-Nt 7 -GFP at the ONM by the co-expression of mRFP-zDHHC11-KASH (Fig. 4e) , suggesting that zDHHC11 equally accepts both Nt 7 constructs as substrate and identifying this enzyme as the major PM-localized PAT for Gαo.
On the other hand, the KASH-fusions of zDHHC2, 5, 8, 14 and 18 showed no effect on the localization of Gαo-Nt 7 -GFP and MGNC-Nt 7 -GFP ( Supplementary Fig. 4d) . Thus, it appears that the predominant PM localization of MGNC-Nt 7 cannot be explained by major differences in substrate specificity of PM-associated zDHHCs.
Next, we applied the SpONM system to Gαo-Nt 7 and MGNC-Nt 7 using the eleven Golgiassociated zDHHCs, as all were efficiently targeted to the ONM by the C-terminal KASHdomain (Fig. 4f,g and Supplementary Fig. 5a,b) . Notably, the mRFP-zDHHC-KASH constructs for zDHHC3 and 7 caused a strong ONM accumulation of Gαo-Nt 7 -GFP ( Fig.   4f,g) . No other zDHHCs showed any activity toward Gαo-Nt 7 -GFP ( Supplementary Fig. 5a) , suggesting a high degree of specificity in substrate recognition among Golgi-associated PATs and identifying zDHHC3 and 7 as the major Golgi-localized PATs for Gαo. MGNC-Nt 7 -GFP was, however, only slightly recruited to the ONM by the KASH-fusions of zDHHC3 and 7 (Fig. 4f,g) , and completely absent from the ONM by the co-expression of the remaining Golgi zDHHC-KASH constructs (Supplementary Fig. 5b) . Quantification showed that Gαo-Nt 7 -GFP accumulates at the ONM roughly 2.0 and 2.5 folds by the co-expression of zDHHC3 and zDHHC7, respectively (Fig. 4h,i) . Conversely, MGNC-Nt 7 -GFP presented a significantly lower ONM recruitment of ~1.5 folds by both zDHHC3 and 7 (Fig. 4h,i) .
To confirm that the PAT activity was responsible for the mislocalization of substrates in the SpONM system, we generated zDHHC-inactive constructs by a Cys-to-Ser mutation in the catalytic DHHC domain 37 . As anticipated, the ability of the KASH-fusions of zDHHC3, 7 and 11 to induce ONM accumulation of Gαo-Nt 7 and MGNC-Nt 7 was completely abolished when DHHS-mutants were co-expressed (Fig. 5a) . Together, our data suggest that the distinct steady-state localizations of Gαo-Nt 7 and MGNC-Nt 7 are due to the differential substrate specificities of PM-and Golgi-localized zDHHCs. This notion implies that the localization of Nt 7 constructs might be shifted toward the PM or Golgi by manipulating the expression level of specific zDHHCs. To test this hypothesis, we co-transfected N2a cells with Gαo-Nt 7 -GFP and HA-zDHHC11, and determined its relative PM content and co-localization with the MannII-BFP Golgi marker. We also co-expressed MGNC-Nt 7 -GFP together with HA-zDHHC3 or HA-zDHHC7. Remarkably, the PM localization of Gαo-Nt 7 strongly increased by the overexpression of zDHHC11, whereas its co-localization with MannII-BFP showed a significant decrease (Fig. 5b,e,f) . On the other hand, MGNC-Nt 7 presented a higher colocalization with the Golgi marker but a significant decrease in PM content by the coexpression of zDHHC3 or zDHHC7 (Fig. 5c-f ). The effects of the different zDHHCs on the steady-state localization of Gαo-Nt 7 and MGNC-Nt 7 were not evident upon co-expression of the corresponding DHHS-mutants ( Fig. 5b-f ), since both Nt 7 constructs showed similar localization patterns as described above (Figs. 1b, 2a-c) . Collectively, these data confirm the notion that local S-palmitoylation plays a major role in the compartmentalization of Gαo-Nt 7 and MGNC-Nt 7 . As the KASH-fusion of zDHHC11 was able to target both Gαo-Nt 7 and MGNC-Nt 7 to the ONM (Fig. 4c-e) , we asked whether it might not discriminate among different Nt 7 substrates. We challenged this notion and found an interesting selectivity for closely related Nt 7 sequences: mRFP-zDHHC11-KASH induced a strong ONM recruitment of MGSSCSR-Nt 7 -GFP but showed a much weaker effect on MGLLCSR-Nt 7 -GFP ( Supplementary Fig. 6a,b) , both sequences commonly found in plant Gα subunits (Supplementary Table 1 ). Thus, it appears that zDHHC3, 7 and 11 can differentiate among Nt 7 substrates with slight variations in amino acid composition.
We then asked whether the higher PM targeting of Gαo-Nt 31 compared to Gαo-Nt 7 might also be the outcome of different zDHHC substrate specificities. Thus, we tested if zDHHC3, 7, and 11 target Gαo-Nt 31 to the ONM with the same efficiency than Gαo-Nt 7 . Although the three zDHHC-KASH constructs relocalized Gαo-Nt 31 -GFP to the ONM, zDHHC3 and zDHHC7 showed a weaker effect when compared to Gαo-Nt 7 -GFP (Fig. 6a,b) . Therefore, the weaker activity of the Golgi zDHHC3 and 7 toward Gαo-Nt 31 predicts an increase in its PM accumulation upon time.
Finally, we analyzed if the SpONM system might reveal local S-plamitoylation of the fulllength Gαo. Thus, we expressed Gαo-GFP together with the KASH-fusions of zDHHC3, 7, and 11, as they showed activity toward Gαo-Nt 7 . Remarkably, full-length Gαo-GFP was targeted to the ONM by all zDHHC constructs (Fig. 6c) . On the other hand, the Cys4 mutant of Gαo, MGNC-Gαo-GFP, localized at the ONM primarily when the KASH-fusion for zDHHC11 was co-expressed ( Supplementary Fig. 6c) , similar to what we observed for MGNC-Nt 7 -GFP (Fig. 4d,f,g) . Overall, our SpONM system shows that local S-palmitoylation 14 of Gαo can cause its in-situ accumulation, indicating that zDHHC selectivity and subcellular localization are key players in the substrate compartmentalization.
That Gα subunits can be found at the Golgi apparatus is known for almost 30 years 45 . The Golgi localization of Gα subunits was assumed to be part of their trafficking pathway to the cell periphery, although some non-canonical functions of G-proteins were described at compartments other than the PM 46 . Only recently, the Golgi-pool of Gα subunits has been implicated in the downstream signaling of typical GPCRs 4 and the KDEL receptor 5, 6 . Thus, subcellular compartmentalization has emerged as an important player in G protein functions.
The present study provides an in-depth characterization of key elements controlling Nmyristoylation, S-palmitoylation and localization of Gαo. More importantly, we developed a system -SpONM -for the rapid detection of zDHHC-substrate pair relations in intact cells, opposing the disruptive biochemical and proteomic techniques currently used to study Spalmitoylation 39, 40 . Altogether, this work supports a model in which local S-palmitoylation by distinct zDHHCs is crucial for Gαo compartmentalization, and most likely for other PMPs (Fig. 6d ). In this model, N-myristoylation allows the nascent protein to reach virtually the whole endomembrane system. Subsequently, specific PM-and Golgi-localized zDHHCs catalyze S-palmitoylation of PMPs, which in turn accumulate at these compartments. Our model is in agreement with the local S-palmitoylation described for substrates such as PSD95 at dendritic spines 38 and calnexin at the ER 47 , but adds the Golgi as a compartment in which PMPs can be locally modified and retained. To our knowledge, S-palmitoylation via the large number of Golgi-localized zDHHCs has been exclusively discussed in the context of Golgi-to-PM transport of integral 48 and PMPs 13 . We hereby challenge the current model and show instead that PMP localization to one compartment or another (including Golgi and PM) is not a consequence of sequential trafficking of the PMPs but rather a direct localization driven by the substrate-specific PAT activities at different compartments.
The fact that zDHHCs can modify multiple substrates has led to the notion that Spalmitoylation is nonspecific and proximity-based. Accordingly, a recent study demonstrated the stochastic S-palmitoylation of engineered Cys in membrane proteins, proposing that catalysis is determined by substrate accessibility and not specific sequence motifs 49 . Several zDHHCs, however, contain PDZ-binding motifs, SH3 domains or ankyrin-repeats outside the catalytic DHHC core that have been implicated in enzyme-substrate interactions 12 .
Importantly, our study uncovered a striking substrate specificity for several zDHHCs toward peptides as small as Gαo-Nt 7 . We additionally showed that minor sequence modifications might result not only in drastic changes in the substrate recognition by zDHHCs, but also in the substrate localization (Fig. 6d) . Another interesting implication of this model is that cells and tissues could shift the localization -and associated function -of PMPs toward one specific compartment or another by controlling the expression of zDHHCs. The large number of zDHHC genes and their distinct tissue-specific expression patterns in humans is thus not surprising 35 . This property might be widespread among Metazoa as Nt 7 constructs showed remarkably similar localization in Drosophila and mammalian cells, and Drosophila contains also a large number of zDHHC genes that are differentially expressed in embryonic and adult tissues 50 . Moreover, it is tempting to speculate that clade-specific zDHHC specializations might account for the distinct Cys position in eukaryotic Gα subunits, especially for Cys at position 5 that dominates in plants and amoebae but is almost absent in Metazoa.
The SpONM system we developed in this study relies on tagging zDHHCs at their Cterminus with the KASH domain of syne-1/nesprin-1 for the ectopic ONM targeting. We applied this strategy only to PM-and Golgi-localized zDHHCs but it might also work for the remaining PATs, excluding zDHHC24 with a predicted C-terminus facing the lumen 40 . Some of the ER-localized zDHHCs, however, showed a prominent ONM localization that might hinder their usage in the SpONM system. In this case, alternative tags for the targeting of ER-localized zDHHCs to the PM, Golgi or mitochondria might be applicable. The C-terminal domain of zDHHCs contains two non-catalytic TTxE and PaCCT (palmitoyltransferase conserved C-terminus) motifs that seem relevant for the overall protein structure 51 . The extreme C-terminal region of zDHHCs appears unstructured and is most likely not involved in the binding and presentation of substrates 12 , implying that the fusion of a KASH domain does not interfere with the substrate specificity. Accordingly, our SpONM pointed to zDHHC3, 7, and 11 as the major PATs for Gαo. This result is in agreement with a previous study showing zDHHC3 and 7 as the main PATs for Gαi2, Gαq and Gαs 52 , although zDHHC11 activity toward Gα subunits has not been detected. This discrepancy could be explained by a differential turnover rate of PM-and Golgi-localized Gα subunits. For instance, if the turnover of the PM pool of Gαi2 is much faster than its Golgi pool, the S-palmitoylation of Gαi2 by the PM-localized zDHHC11 might have gone unnoticed by the biochemical methods used in that study. The SpONM system might therefore provide an advantage in sensitivity by targeting zDHHCs to the ONM, as even small amounts of a substrate are visible at the ectopic compartment.
A polybasic stretch present in the N-terminal region of all Gα subunits has been implicated in PM binding 53 . The Gαo-Nt 31 construct containing the basic region within the α-helix showed indeed a greater steady-state localization at the PM compared to Gαo-Nt 7 . In the SpONM system, the presence of the α-helix in Gαo-Nt 31 appears to impact substrate recognition by the Golgi-associated zDHHC3 and 7, but not by the PM-localized zDHHC11. Thus, these data point out to a second level of complexity in substrate affinity by zDHHCs, where regions distant from the palmitoylatable Cys might subserve regulatory functions. How the polybasic region in Gαo impacts substrate specificity by zDHHCs remains to be clarified.
S-palmitoylation has gained much attention lately as a potential target for drug discovery to combat pathologies mediated by proteins such as the oncogenic Ras and infectious diseases like malaria caused by Plasmodium falciparum or the respiratory syndromes caused by coronaviruses 54-56 . Our SpONM system could enable screens for molecules that perturb siteand zDHHC-specific S-palmitoylation of pathological proteins. This approach may provide an attractive alternative to directly target the desired oncogene (or protein of another pathogenic activity), focusing instead on developing tools to target the zDHHCs controlling the proper (or pathological) localization of the protein. In this regard, zDHHCs can be targeted by the drug discovery efforts. The large number of these enzymes in humans and their distinct expression patterns provide the reasoning to expect the power of such an approach in obtaining the favorable pharmacodynamics with limited side effects. Another approach might aim at targeting the specific zDHHC-substrate interaction pairs; this avenue is expected to deliver even more specific targeted therapies.
To sum up, we have delineated the code describing the Golgi vs. PM localization of the G protein Gαo, showed that these two localizations are independent from the traditionally assumed Golgi-to-PM trafficking nor preferential interactions with major lipids, and discovered a universal mechanism controlling PMP localization to its target membrane through substrate selectivity of differently localized PATs. This major insight into the cell biology of protein subcellular localization may pave the way to novel drug discovery principles.
Monoclonal antibodies (mAbs) against flotillin-2 (Cat# 610383) and GM130 (Cat# 610823) were from BD Biosciences, mAb against HA (Cat# 11867423001) Male Drosophila Schneider-2 (S2) cells were maintained in Schneider's Drosophila Medium (Lonza) supplemented with 10% FCS and 1% penicillin-streptomycin at 28°C.
All vector transfections were carried out with X-tremeGENE 9 or HP (Roche) according to manufacturer's instructions.
The plasmids Gαo-GFP, GalT-GFP, and GalT-mRFP for Drosophila expression were previously described 6 . To generate the GFP-fusion of Gαo-Nt 7 and Gαo-Nt 31 , the fragments were PCR-amplified (primers listed in Supplementary Table 3) from Gαo-GFP, products were cut with KpnI/AgeI and cloned in frame into the same sites of pEGFP-N1 (Clontech). The Gαo-Nt 8-31 -GFP sequence was PCR-amplified from Gαo-Nt 31 -GFP, the product cut with KpnI/NotI and used to replace the GFP sequence in pEGFP-N1 cut with the same enzymes.
Gαo-Nt 7 -GFP was used as template for the PCR amplification of the following All fluorescent-labelled secondary antibodies were from Jackson ImmunoResearch.
Coverslips were finally mounted with Vectashield on microscope slides. Cells were recorded with a Plan-Apochromat 63x/1.4 oil objective on a LSM800 Confocal Microscope and further processed using the ZEN blue software (all Zeiss). When required (see below), mean fluorescence intensity was determined from confocal images using ImageJ (National Institutes of Health).
N2a cells expressing the GFP-fusion constructs were immunostained as indicated above using a mouse monoclonal antibody (mAb) against GM130 to visualize the Golgi apparatus, and a DAPI staining for the nucleus (both not shown in all final images). To avoid interferences due to different expression levels of GFP-constructs among cell population, mean fluorescence intensity was measured at the GM130-positive Golgi region as well as at the total cell area, and ratio values were used to determine the relative Golgi accumulation of the constructs. Simultaneously, mean fluorescence intensity was determined at an unbroken region of the PM lacking membrane protrusions, and the ratio over total cell fluorescence was used to define relative PM content for each GFP-construct. One-way ANOVA or Student's t-test were used for statistical analysis of ≥50 cells per condition and from at least 3 independent experiments.
N2a cells were transfected and immunostained against GM130 as described above. To determine co-localization of the various GFP constructs and the GM130 signal, an area covering the whole perinuclear region but excluding the PM was selected in confocal images and the Pearson's correlation was calculated using the co-localization tool of ImageJ. The same analysis was done in N2a cells co-expressing the phosphatidylserine biosensor mRFP-Lact-C2 27 and the different GFP-fusions, transfected at equal plasmid ratio. The Golgi marker
GalT-GFP was used as control. This analysis was also done in N2a cells co-transfected with GFP-fusions, HA-tagged zDHHCs and MannII-BFP as the Golgi marker (2:4:1 plasmid ratio).
One-way ANOVA test was used for statistical analysis of >30 cells per condition.
The recombinant GST-tagged Nanobody against GFP 64 
Rosettagami (Novagen) was purified with glutathione Sepharose 4B beads according to manufacturer's instructions. Protein purity was assessed by SDS-PAGE and Coomassie blue staining.
N2a cells were co-transfected with the different Gαo-GFP constructs and mRFP-Gβ1/Gγ3 at 1:1:1 plasmid ratio. After 24 hr transfection, cells were resuspended with ice-cold GST-lysis buffer (1% Triton X-100 and 10% glycerol in PBS) supplemented with a protease inhibitor cocktail (Roche) and passed 10 times through a 25G needle. Extracts were cleared by centrifugation at 15,000xg for 20 min at 4°C, and supernatants were incubated with 1 µg of purified GST-tagged GFP-Nanobody for 30 min on ice. Then, 20 µL of Glutathione Sepharose 4B beads were added and samples rotated overnight at 4°C. Beads were repeatedly washed with GST-lysis buffer, prepared for SDS-PAGE, and finally analyzed by Western blot using a rabbit polyclonal antibody (pAb) against GFP and a mouse mAb against mRFP. HRP-conjugated secondary pAbs (Jackson ImmunoResearch) were used for enhanced chemiluminescence (ECL) detection. The co-immunoprecipitation was done in duplicate with very similar outcomes.
N2a cells were transfected for 24 hr and cell extracts were prepared using non-denaturing conditions and fractionated by high-speed centrifugation as previously described 62 . Briefly, cells were rinsed twice with PBS and resuspended in hypo-osmotic buffer (20 mM Hepes, pH 7.0) supplemented with a protease inhibitor cocktail. The cell suspension was passed 20 times through a 25G needle, and nuclei and unbroken cells were removed by centrifugation at 700xg for 10 min at 4°C. The supernatant was centrifuged at 100,000xg for 60 min at 4°C.
The new supernatant (cytosolic fraction) was directly prepared for SDS-PAGE, and the pellet (membrane fraction) was gently washed with hypo-osmotic buffer and resuspended in GSTlysis buffer supplemented with 0.5% SDS and protease inhibitors. The membrane fraction was cleared by centrifugation at 15,000xg for 20 min at 4°C, and prepared for SDS-PAGE.
Western blot analysis was done as above using the pAb against GFP and the mouse mAbs against flotillin-2 and GAPDH as endogenous membrane and cytosolic markers, respectively. Two-way ANOVA was used for statistical analysis of 4-6 independent experiments.
After 24 hr of transfection, N2a cells were washed twice with TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 10% glycerol) and cell extracts were prepared as explained above in TBS supplemented with 0.5% Tx-100 and protease inhibitors. Membrane lipid strips were blocked in 3% BSA in TBS supplemented with 0.1% Tween-20 (TBS-T) for 1 hr at 4°C. Then, strips were incubated overnight at 4°C with cleared cell extracts previously diluted 1/10 in TBS-T supplemented with 1% BSA. Strips were incubated first with a pAb against GFP, and then with a secondary HRP-conjugated pAb to detect GFP-constructs bound to lipid dots. The protein-lipid overlay assay was done in duplicate for each condition with very similar results.
To identify Gα subunit paralogs in eukaryotic genomes, the full-length amino acid sequence of Gαo was used for search queries in the NCBI protein Blast browser (blast.ncbi.nlm.nih.gov/Blast.cgi). Specifically, species with sequenced genomes were selected from Ensembl databases (ensembl.org) for vertebrates, plants, fungi, metazoa and protist. For the remaining phylogenetic clades, Gα paralogs were found as above and using the NCBI Taxonomy browser (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) as guide for clade classification. Sequences were manually curated and when annotated as partial were not considered further for this analysis. A total of 262 species were found to express Gα paralogs among the clades Amoebozoa, Discoba, Haptista, Metamonada, Opisthokonta, Sar, and Viridiplantae (Table S1 ). Logo representation of the unique sequences selected for the different Gαo-Nt 7 categories (Table S2) were generated from stacks using ImageJ, and the mean fluorescence intensity of an area at the center of the MannII-mRFP Golgi region was measured from stacks in >10 cells per condition.
S-palmitoylation at the outer nuclear membrane (SpONM) HeLa cells were co-transfected as above with plasmids encoding the GFP-fusion, SUN2, and control mRFP-KASH or mRFP-zDHHC-KASH at a 1:3:3 plasmid ratio. Cells were then seeded on poly-L-lysine-coated coverslips, fixed and prepared for microscopy. For quantification, mean GFP-fluorescence intensity at the ONM region labelled by the mRFP-KASH fusion was determined using ImageJ. Simultaneously, GFP-fluorescence was measured at a nearby cytosolic region as well, and the ratio ONM over cytosol was used to define the relative ONM content of each GFP-construct. One-way ANOVA or Student's t-test were used for statistical analysis of ≥40 cells per condition and from at least 3 independent experiments. Selected areas are magnified to the right. Quantification of mean fluorescence intensity ratios