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• W2900528571 abstract "•DHHC S-palmitoyltransferases are enriched in the cis-Golgi•S-palmitoylation induces concentration of membrane cargo at the cisternal rim•The rate of anterograde transport across the Golgi is controlled by S-palmitoylation 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. 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. What are the basic principles of cargo selection for anterograde transport in the Golgi stack? Thirty years ago, we first reported that palmitoyl (C16)-coenzyme A (coA) stimulated intra-Golgi transport in a cell-free extract (Glick and Rothman, 1987Glick B.S. Rothman J.E. Possible role for fatty acyl-coenzyme A in intracellular protein transport.Nature. 1987; 326: 309-312Crossref PubMed Scopus (209) Google Scholar). Removing palmitoyl-CoA from the extract revealed a strong requirement for acyl-CoA, which was met only by palmitoyl-CoA and not C14 or C18 acyl-CoAs. Transport was inhibited by adding a non-hydrolyzable analog of palmitoyl-CoA, confirming that acyl chain transfer to an acceptor was required. Remarkably, the assembly of COPI-coated vesicles from the Golgi cisternal rims, containing the cargo protein VSV G, was revealed to be strongly dependent on this palmitoylation event (Pfanner et al., 1989Pfanner N. Orci L. Glick B.S. Amherdt M. Arden S.R. Malhotra V. Rothman J.E. Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae.Cell. 1989; 59: 95-102Abstract Full Text PDF PubMed Scopus (165) Google Scholar). The molecular basis of this striking and fundamental finding has remained obscure since then. Now, as a result of important progress in understanding the enzymes that mediate palmitoylation (Fukata et al., 2004Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, Lobo et al., 2002Lobo S. Greentree W.K. Linder M.E. Deschenes R.J. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.J. Biol. Chem. 2002; 277: 41268-41273Crossref PubMed Scopus (364) Google Scholar, Roth et al., 2002Roth A.F. Feng Y. Chen L. Davis N.G. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.J. Cell Biol. 2002; 159: 23-28Crossref PubMed Scopus (375) Google Scholar, Politis et al., 2005Politis E.G. Roth A.F. Davis N.G. Transmembrane topology of the protein palmitoyl transferase Akr1.J. Biol. Chem. 2005; 280: 10156-10163Crossref PubMed Scopus (69) Google Scholar) and advances allowing sites of palmitoylation to be visualized by light microscopy (Kolb et al., 2001Kolb H.C. Finn M.G. Sharpless K.B. Click chemistry: diverse chemical function from a few good reactions.Angew. Chem. Int. Ed. 2001; 40: 2004-2021Crossref PubMed Scopus (11474) Google Scholar), as well as broad advances in molecular cell biology, this investigation is now in a position to be reopened. A large variety of transmembrane and soluble proteins are modified with palmitate by its attachment onto cysteine residues of proteins via a thioester linkage, which strikingly can alter the biophysical properties of the acylated proteins (Smotrys and Linder, 2004Smotrys J.E. Linder M.E. Palmitoylation of intracellular signaling proteins: regulation and function.Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (479) Google Scholar). In most cases, these sites of acylation are retained, while in other cases, particularly for soluble proteins involved in signaling cascades, a highly dynamic turnover is observed (Fukata and Fukata, 2010Fukata Y. Fukata M. Protein palmitoylation in neuronal development and synaptic plasticity.Nat. Rev. Neurosci. 2010; 11: 161-175Crossref PubMed Scopus (426) Google Scholar, Linder and Deschenes, 2007Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (750) Google Scholar, Demers et al., 2014Demers A. Ran Z. Deng Q. Wang D. Edman B. Lu W. Li F. .Palmitoylation is required for intracellular trafficking of influenza B virus NB protein and efficient influenza B virus growth in vitro.J. Gen. Virol. 2014; 95: 1211-1220Crossref PubMed Scopus (13) Google Scholar, El-Husseini et al., 2002El-Husseini Ael-D. Schnell E. Dakoji S. Sweeney N. Zhou Q. Prange O. Gauthier-Campbell C. Aguilera-Moreno A. Nicoll R.A. Bredt D.S. Synaptic strength regulated by palmitate cycling on PSD-95.Cell. 2002; 108: 849-863Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, Salaun et al., 2010Salaun C. Greaves J. Chamberlain L.H. The intracellular dynamic of protein palmitoylation.J. Cell Biol. 2010; 191: 1229-1238Crossref PubMed Scopus (233) Google Scholar). Furthermore, S-palmitoylation was shown to induce wide ranging regulatory effects on proteins, including membrane-targeting, protein-protein interactions, protein folding and stability, sorting of soluble palmitoylated proteins to the plasma membrane (Salaun et al., 2010Salaun C. Greaves J. Chamberlain L.H. The intracellular dynamic of protein palmitoylation.J. Cell Biol. 2010; 191: 1229-1238Crossref PubMed Scopus (233) Google Scholar, Linder and Deschenes, 2007Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (750) Google Scholar), and, recently, modulation of membrane protein spontaneous curvature (Chlanda et al., 2017Chlanda P. Mekhedov E. Waters H. Sodt A. Schwartz C. Nair V. Blank P.S. Zimmerberg J. Palmitoylation contributes to membrane curvature in influenza A virus assembly and hemagglutinin-mediated membrane fusion.J. Virol. 2017; 91https://doi.org/10.1128/JVI.00947-17Crossref PubMed Scopus (45) Google Scholar). Currently, proteomic analysis has identified more than 500 proteins as S-palmitoylated, which includes both integral and peripheral membrane proteins (Blanc et al., 2015Blanc M. David F. Abrami L. Migliozzi D. Armand F. Bürgi J. van der Goot F.G. SwissPalm: protein palmitoylation database.F1000Res. 2015; 4: 261Crossref PubMed Scopus (147) Google Scholar). Given the ubiquity of S-palmitoylation and its involvement in key physiological processes, it is no surprise that palmitoylation has been linked to a number of human diseases (Greaves and Chamberlain, 2011Greaves J. Chamberlain L.H. DHHC palmitoyl transferases: substrate interactions and (patho) physiology.Trends Biochem. Sci. 2011; 36: 245-253Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In mammals, S-palmitoylation is catalyzed by a family of 23 acyltransferase enzymes (Fukata et al., 2004Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, Lobo et al., 2002Lobo S. Greentree W.K. Linder M.E. Deschenes R.J. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.J. Biol. Chem. 2002; 277: 41268-41273Crossref PubMed Scopus (364) Google Scholar, Roth et al., 2002Roth A.F. Feng Y. Chen L. Davis N.G. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.J. Cell Biol. 2002; 159: 23-28Crossref PubMed Scopus (375) Google Scholar). These enzymes share a conserved membrane topology with 4–6 transmembrane domains, as well as a prominent DHHC tetrapeptide positioned in a cysteine-rich domain (CRD) that is crucial for acylation activity (Politis et al., 2005Politis E.G. Roth A.F. Davis N.G. Transmembrane topology of the protein palmitoyl transferase Akr1.J. Biol. Chem. 2005; 280: 10156-10163Crossref PubMed Scopus (69) Google Scholar). The DHHC-CRD domain resides on a cytosolic loop, thereby allowing it to access substrates close to the membrane. Palmitoylation by DHHC enzymes is thought to occur via a two-step mechanism in which the DHHC enzymes first form an acyl-enzyme intermediate on cysteine residues (autoacylation) and then subsequently transfer the palmitate to a cysteine residue on the target protein (Jennings and Linder, 2012Jennings B.C. Linder M.E. DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.J. Biol. Chem. 2012; 287: 7236-7245Crossref PubMed Scopus (137) Google Scholar, Mitchell et al., 2010Mitchell D.A. Mitchell G. Ling Y. Budde C. Deschenes R.J. Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes.J. Biol. Chem. 2010; 285: 38104-38114Crossref PubMed Scopus (117) Google Scholar). Recently, the structure of DHHCs 15 and 20 was solved in a lipidic cubic phase, suggesting the orientation of the active site close to the membrane (Rana et al., 2018Rana M.S. Kumar P. Lee C.J. Verardi R. Rajashankar K.R. Banerjee A. Fatty acyl recognition and transfer by an integral membrane S-acyltransferase.Science. 2018; 359 (6372, eaao6326)Crossref PubMed Scopus (116) Google Scholar). Early biochemical and localization studies have concluded that the majority of the DHHC enzymes reside on the endoplasmic reticulum (ER) and the Golgi apparatus (Ohno et al., 2006Ohno Y. Kihara A. Sano T. Igarashi Y. Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.Biochim. Biophys. Acta. 2006; 1761: 474-483Crossref PubMed Scopus (319) Google Scholar). In the present study, we revisit the requirement for palmitoyl-CoA on the sorting and trafficking of protein cargo within the Golgi. We mapped the subcellular localization of the 23 DHHC isoforms and discovered that the majority of Golgi-localized DHHCs are concentrated in the cis-Golgi. By employing a metabolic labeling strategy paired with click chemistry and super-resolution fluorescence and electron microscopy, we confirmed that the bulk of palmitate was incorporated into anterograde cargo into the cis-Golgi. Unexpectedly, we discovered that palmitoylation in the cis-Golgi accelerates the transport of these proteins through the Golgi. Strikingly, the palmitoylated proteins are concentrated in the highly curved rims of the cis-Golgi, which we suggest may explain the observed acceleration in rate. In order to identify the main site of bulk S-acylation in HeLa cells, a metabolic labeling strategy was combined with a click chemistry-based approach (Kolb et al., 2001Kolb H.C. Finn M.G. Sharpless K.B. Click chemistry: diverse chemical function from a few good reactions.Angew. Chem. Int. Ed. 2001; 40: 2004-2021Crossref PubMed Scopus (11474) Google Scholar). HeLa cells were labeled with different molecular species of fatty acid analogs, fixed, and subjected to a copper-dependent azide-alkyne cycloaddition (CuAAC) to azide-Alexa 647. Only the C16 and C18 probes efficiently labeled the Golgi, but not the C14 analog, while lipid-droplet-like structures were observed in each case (Figures 1A and S1A). Importantly, labeling of the Golgi with the C16 probe required activation with CoA (sensitive to Triacsin C [TriaC], inhibitor of the long chain fatty acid CoA synthetase; Omura et al., 1986Omura S. Tomoda H. Xu Q.M. Takahashi Y. Iwai Y. Triacsins, new inhibitors of acyl-CoA synthetase produced by Streptomyces sp.J. Antibiot. 1986; 39: 1211-1218Crossref PubMed Scopus (64) Google Scholar), catalysis by DHHC palmitoyltransferases (sensitive to 2-bromopalmitate [2-BP], inhibitor of DHHCs; Coleman et al., 1992Coleman R.A. Rao P. Fogelsong R.J. Bardes E.S.G. 2-bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes.Biochim. Biophys. Acta. 1992; 1125: 203-209Crossref PubMed Scopus (80) Google Scholar), and the presence of biosynthetic cargo at the Golgi (sensitive to cycloheximide [CHX]-induced purge of the Golgi; Taylor et al., 1984Taylor J.A. Limbrick A.R. Allan D. Judah J.D. Isolation of highly purified Golgi membranes from rat liver. Use of cycloheximide in vivo to remove Golgi contents.Biochim. Biophys. Acta. 1984; 769: 171-178Crossref PubMed Scopus (18) Google Scholar, Todorow et al., 2000Todorow Z. Spang A. Carmack E. Yates J. Schekman R. Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2000; 97: 13643-13648Crossref PubMed Scopus (43) Google Scholar), altogether supporting that the majority of signal observed stemmed from S-palmitoylated proteins (Figures 1B and S1B–S1D). Furthermore, the use of C16 and C18 probes coincided with the appearance of distinct protein bands after SDS-PAGE and in-gel fluorescence, which, as expected of S-palmitoylated proteins, were hydroxylamine sensitive (Figure S1E), cleaving S-linked acyl bonds rather than O-linked acyl bonds (Magee et al., 1984Magee A.I. Koyama A.H. Malfer C. Wen D. Schlesinger M.J. .Release of fatty acids from virus glycoproteins by hydroxylamine.Biochim. Biophys. Acta. 1984; 798: 156-166Crossref PubMed Scopus (70) Google Scholar). We next quantified the co-localization of the (C16) palmitate probe at different times of labeling from confocal z stacks with regard to endogenous cis- and trans-Golgi markers, the ER, and lipid droplets (Figure S1F). The majority of the observed signal over the course of the pulse originated from the Golgi and lipid droplets. 3D structured illumination super-resolution microscopy (SIM) revealed that within the Golgi, the main site of incorporation was in the cis-compartment (Figures 1A and 1C). Within the next 10 min of a “chase” experiment, in which an excess of natural palmitic acid was added to largely replace the analog from that time onward, co-localization with the cis-Golgi declined while increasing in the trans-Golgi (with no appearance of the probe in the ER; Figures 1C and S1G). This would be expected if the bulk of labeled proteins was anterograde-directed cargo. Consistent with this finding, the bulk of the protein at the Golgi exited within 20 min, as revealed by quantifying several hundreds of cells in a “chase” experiment (Figures 1D and S1H). Palmitoylation of Cysteine residues in membrane proteins occurs at juxta-membrane positions, with more than 500 proteins previously identified via proteomic analyses (Blanc et al., 2015Blanc M. David F. Abrami L. Migliozzi D. Armand F. Bürgi J. van der Goot F.G. SwissPalm: protein palmitoylation database.F1000Res. 2015; 4: 261Crossref PubMed Scopus (147) Google Scholar). Several DHHC palmitoyltransferases are localized within the Golgi (Ohno et al., 2006Ohno Y. Kihara A. Sano T. Igarashi Y. Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.Biochim. Biophys. Acta. 2006; 1761: 474-483Crossref PubMed Scopus (319) Google Scholar). These enzymes generally accept C16 (palmitate) and to a lower extent C18 (stearate), but not shorter (C14, myristate) fatty acids as coA-activated substrates (Politis et al., 2005Politis E.G. Roth A.F. Davis N.G. Transmembrane topology of the protein palmitoyl transferase Akr1.J. Biol. Chem. 2005; 280: 10156-10163Crossref PubMed Scopus (69) Google Scholar, Jennings and Linder, 2012Jennings B.C. Linder M.E. DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.J. Biol. Chem. 2012; 287: 7236-7245Crossref PubMed Scopus (137) Google Scholar, Mitchell et al., 2010Mitchell D.A. Mitchell G. Ling Y. Budde C. Deschenes R.J. Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes.J. Biol. Chem. 2010; 285: 38104-38114Crossref PubMed Scopus (117) Google Scholar). S-palmitoylation in the cis- prior to the trans-Golgi suggested the presence of distinct DHHCs in the cis-Golgi. In order to identify DHHC isoform candidates that exclusively localize to the Golgi, the subcellular localization of a mouse ortholog library of all 23 DHHCs was mapped (Fukata et al., 2004Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Indeed, 17 of the 23 DHHCs exhibited a partial Golgi localization, with 11 predominantly localizing to the Golgi (Figures S2A–S2C). The corresponding human orthologs were cloned as aminoterminal fusion proteins to the SNAP-tag protein, and the intra-Golgi localization of the human DHHC candidates was then probed with endogenous cis- and trans-Golgi markers using confocal microscopy (Figures 1F and S2D). While the human DHHC 16 and 18 localized to the ER and PM, respectively, the remaining 9 candidates exhibited an asymmetrical distribution across the cis-to-trans Golgi axis: DHHCs 3, 7, 13, 17, 21, and 22 specifically localized to the cis. zDHHCs 9, 11, and 15 localized partially to the trans-Golgi, and to differing extents, to post-Golgi structures (Figures 1G and S2D). To determine which Golgi-localized S-palmitoyltransferases contribute to the observed incorporation of palmitate analog into anterograde cargo in the cis-Golgi (Figure 1C), each Golgi candidate (human) DHHC enzyme was overexpressed (Figures 2A and S2E). DHHC3 and DHHC7 dramatically increased both the rate of incorporation as well as the rate of Golgi exit when chased with natural palmitate (Figures S3A and S3B). Analogous to untransfected cells, DHHC-catalyzed S-palmitoylation at the Golgi required activation with CoA, DHHC enzyme function, and the presence of biosynthetic cargo (Figures 2B and S1B–S1D). Furthermore, the increased incorporation of palmitate in the Golgi area resulted from acyl transfer since enzymatically inactive versions of DHHC3 and DHHC7 (DHHS) did not increase the incorporation (Figures 2C, S3C, and S3D). As expected, the palmitate analog incorporated by both DHHC3 and DHHC7 was released from fixed, permeabilized cells upon exposure to hydroxylamine (Figures 2C and S1B). Consistent with the initial concentration of basal alkyne-palmitate into the cis-Golgi (Figures 1A and 1C), DHHC3 and DHHC7 are localized to cis- but not trans-Golgi regions when co-localized with endogenous markers using 3D-SIM (Figures 1E and S3E). DHHC7 was recently mapped to the trans-Golgi, but by employing overexpressed Golgi markers (Du et al., 2017Du K. Murakami S. Sun Y. Kilpatrick C.L. Luscher B. DHHC7 Palmitoylates glucose transporter 4 (Glut4) and regulates Glut4 membrane translocation.J. Biol. Chem. 2017; 292: 2979-2991Crossref PubMed Scopus (34) Google Scholar). In fact, we noticed that strong DHHC overexpression results in the disappearance of endogenous Golgi markers (Figure S3F) and conclude that the localization of these DHHCs within the stack can only be accurately mapped in the presence of these endogenous markers (at low DHHC expression levels). The closely related DHHCs 3 and 7 are ubiquitously expressed (Ohno et al., 2006Ohno Y. Kihara A. Sano T. Igarashi Y. Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.Biochim. Biophys. Acta. 2006; 1761: 474-483Crossref PubMed Scopus (319) Google Scholar), and various studies revealed that overexpression of the corresponding mouse orthologs efficiently catalyzed S-palmitoylation of a broad variety of substrates (Gottlieb et al., 2015Gottlieb C.D. Zhang S. Linder M.E. The cysteine-rich domain of the DHHC3 palmitoyltransferase is palmitoylated and contains tightly bound zinc.J. Biol. Chem. 2015; 290: 29259-29269Crossref PubMed Scopus (37) Google Scholar, Greaves et al., 2017Greaves J. Munro K.R. Davidson S.C. Riviere M. Wojno J. Smith T.K. Tomkinson N.C. Chamberlain L.H. Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.Proc. Natl. Acad. Sci. USA. 2017; 114: E1365-E1374Crossref PubMed Scopus (82) Google Scholar). DHHC3 and DHHC7 protein substrates consist of numerous distinct proteins visible in SDS-PAGE that are sensitive to hydroxylamine cleavage (Figure 2D). As the rapid Golgi exit of S-palmitoylated proteins (with lack of rising palmitate levels in the ER) implied that the majority of DHHC substrates are anterograde cargo, putatively plasma membrane residents, bioinformatical predictions on compartment-specific datasets were performed (ER, Golgi, PM). We compared (1) the total fraction of proteins containing high-confidence S-palmitoylation sites, (2) the average number of predicted sites per protein, and (3) the fraction of proteins containing signal sequences, cytoplasmic Cys residues, and high-confidence S-palmitoylation sites (Figures S3G and S3H), supporting that indeed anterograde cargo are a major class of DHHC substrates, as all values peak in the PM dataset. We therefore metabolically labeled cells expressing either DHHC3, 7, or their enzymatically inactive variants (DHHS) with alkyne-palmitate for 10 min, chased the probe for different times, and after fixation and permeabilization, performed total internal reflection fluorescence (TIRF) microscopy. Strikingly, overexpressing either DHHC3 or DHHC7 (Figures 2E, 2F, S4A, and S4B) markedly accelerated the rate and extent of appearance of S-palmitoylated proteins at the cell surface (3-fold after 60 min). Furthermore, this acceleration required active DHHC forms of the enzymes, with no acceleration observed with DHHS point mutants. To confirm transit of the anterograde palmitoylated cargo, a strategy was employed that would allow for transmission electron microscopy of palmitoylated proteins. HeLa cells were stably transfected with SNAP-DHHC3 to catalyze the incorporation of alkyne-palmitate into the Golgi (Figure S4C). Our previous approach was adapted by employing azide-biotin as a ligand for alkyne-palmitate, which was then detected using a fluorogold-labeled (containing 1.4 nm nanogold and Alexa488) Streptavidin, allowing the use of light and electron microscopy (Figure S4D). We again performed a time-course experiment to monitor the distribution of the probe within intracellular membranes over time, and its enrichment was quantified in the Golgi. In agreement with the partitioning of alkyne-palmitate observed through the Golgi on the light level (Figures 1 and 2), an extensive labeling of Golgi membranes after 30 min of pulse was confirmed (Figure S4E). After 60 min, a marked labeling of the plasma membrane concurrent with a reduction of membrane labeling within the Golgi was observed (Figures S4E and S4F). The marked increase in the amount of S-palmitoylated protein detected at the plasma membrane upon overexpression of DHHC3 or 7 (in the cis-Golgi) prompted the hypothesis that palmitoylation accelerates trafficking of anterograde cargo at the level of the Golgi. There is some precedent as palmitoylation at the Golgi had been established as an important step in the vectorial transport of multiple soluble proteins, e.g., H-Ras, CSP, and SNAP25 (Gonzalo and Linder, 1998Gonzalo S. Linder M.E. SNAP-25 palmitoylation and plasma membrane targeting require a functional secretory pathway.Mol. Biol. Cell. 1998; 9: 585-597Crossref PubMed Scopus (156) Google Scholar, Greaves et al., 2008Greaves J. Salaun C. Fukata Y. Fukata M. Chamberlain L.H. .Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein.J. Biol. Chem. 2008; 283: 25014-25026Crossref PubMed Scopus (93) Google Scholar, Apolloni et al., 2000Apolloni A. Prior I.A. Lindsay M. Parton R.G. Hancock J.F. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway.Mol. Cell. Biol. 2000; 20: 2475-2487Crossref PubMed Scopus (345) Google Scholar). For such soluble proteins, palmitoylation acts as a post-ER membrane-targeting signal, restricting peripheral protein function to the Golgi and PM, while enabling their release via thioesterases. Further, a plethora of integral membrane proteins has been identified as being S-palmitoylated, including most viral spike proteins, plasma membrane channels, and receptors (Schmidt and Schlesinger, 1979Schmidt M.F. Schlesinger M.J. Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein.Cell. 1979; 17: 813-819Abstract Full Text PDF PubMed Scopus (181) Google Scholar, Chien et al., 1996Chien A.J. Carr K.M. Shirokov R.E. Rios E. Hosey M.M. Identification of palmitoylation sites within the L-type calcium channel β2a subunit and effects on channel function.J. Biol. Chem. 1996; 271: 26465-26468Crossref PubMed Scopus (162) Google Scholar, Charollais and Van Der Goot, 2009Charollais J. Van Der Goot F.G. Palmitoylation of membrane proteins (review).Mol. Membr. Biol. 2009; 26: 55-66Crossref PubMed Scopus (133) Google Scholar). Would palmitoylation of individual anterograde (integral) membrane protein cargo impact their trafficking, as we observed for the bulk of palmitoylated proteins? To investigate this in more detail, we compared the rate of anterograde transport of two well-characterized model palmitoylated cargo proteins (VSV G protein and transferrin receptor) compared to non-palmitoylated mutants in which the attachment site cysteines were mutated to alanines (VSVG-C490A, TfR-C62AC67A). S-palmitoylation for both model proteins depended on the presence of Cys and their release from the ER (Figures S5A and S5H). For VSV G, a wave of anterograde cargo can be generated by employing a temperature-sensitive mutant (tsO45, Bergmann, 1989Bergmann J.E. Using temperature-sensitive mutants of VSV to study membrane protein biogenesis.Methods Cell Biol. 1989; 32: 85-110Crossref PubMed Scopus (88) Google Scholar) that under the non-permissive temperature (40.5°C) is blocked in the ER and can be released at 32°C. Employing a surface biotinylation strategy, we observed a marked difference in the amount detected on the cell surface 30 min post release from the ER: wt VSV G exhibited 50% increased PM levels, while similar levels of Cys→Ala VSVG were only observed after at 60 min (Figure 3A, upper left panel; Figure S5B). Where did this acceleration occur? Over 30 years ago, Rose and colleagues had compared the rate of acquisition of EndoH resistance carbohydrates between wt and the same Cys→Ala mutant VSV G protein and did not observe any significant differences in these rates (Rose et al., 1984Rose J.K. Adams G.A. Gallione C.J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition.Proc. Natl. Acad. Sci. USA. 1984; 81: 2050-2054Crossref PubMed Scopus (94) Google Scholar). We revisited these experiments, and indeed an identical rate of entry of wt and variant G into the Golgi was observed (Figure 3A, upper right panel; Figure S5C). EndoH-resistance is achieved upon arrival of the cargo in the cis-to-medial Golgi, where the concentrations of alpha-Mannosidases I/II peak, hence monitoring the rate of transport from the ER to the Golgi, but not beyond this point. We therefore speculated that any contribution of S-palmitoylation to ER→PM trafficking was not at the ER→Golgi but either within the Golgi or Golgi/TGN→PM. When performing surface biotinylation experiments in conjunction with a 20°C block (to accumulate cargo in the TGN prior to its release), no differences were observed in surface arrival between the constructs (Figure 3A, lower left panel; Figure S5D), supporting a palmitoylation-dependent acceleration of G protein from the cis- to the trans-Golgi. To test this possibility, the rate of acquisition of galactose was measured in the trans-Golgi by using the terminal galactose residue-specific lectin jacalin. While wt G rapidly became positive for galactose 10 min post release and was fully processed within the following 10 min, in which further extension of the N-glycans masks detection by jacalin, Cys→Ala G protein revealed a significantly delayed peak at 30 min and elevated levels even at 60 min (Figure 3A, lower right panel; Figure S5E). An accelerated arrival of wt G in the trans-Golgi was confirmed by confocal co-localization with endogenous Golgi markers (Figure S5F), excluding the possibility of an altered access to the glycosyltransferase within the trans-Golgi. To assess whe" @default.
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• W2900528571 title "S-Palmitoylation Sorts Membrane Cargo for Anterograde Transport in the Golgi" @default.
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• W2900528571 doi "https://doi.org/10.1016/j.devcel.2018.10.024" @default.
• W2900528571 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6251505" @default.
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