Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.     

A dedicated Wnt secretion factor

The Wnt family of signaling proteins mediates cell-cell communication during development. In this issue of Cell, B?nziger et al. (2006) and Bartscherer et al. (2006) identify Wntless/Evi, a multipass transmembrane protein in the secretory pathway of Wnt-producing cells that promotes Wnt secretion.     

Roles of N-glycosylation and lipidation in Wg secretion and signaling

Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modification at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo receptor involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.     

A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities

Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.     

Wnt Trafficking: New Insights into Wnt Maturation,Secretion and Spreading

Proteins of the Wnt family are secreted signaling molecules that regulate multiple processes in animal development and control tissue homeostasis in the adult. Wnts spread over considerable distances to regulate gene expression in cells located at distant sites. Paradoxically, Wnts are poorly mobile because of their posttranslational modification with lipids. Recent evidence suggests that several pathways exist that are capable of transforming hydrophobic, insoluble Wnts into long‐range signaling molecules. Furthermore, the discovery of Wntless as a protein specifically required for the secretion of Wnt suggests that Wnt trafficking through the secretory pathway is already under special scrutiny. Here, we review recent data on the molecular machinery that controls Wnt secretion and discuss how Wnts can be mobilized for long‐range signaling.     

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.     

A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration

Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration.     

Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.     

Protein kinase CK2 is required for Wntless internalization and Wnt secretion

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.     

The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network

Secreted Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wnt secretion. Recent studies have identified Wntless (Wls) and the retromer complex as essential components involved in Wnt signaling. While Wls has been shown to be essential for Wnt secretion, the function(s) of the retromer complex in Wnt signaling is unknown. Here, we have examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. We provide compelling evidence that the retromer complex is required for Wnt secretion. Importantly, Vps35 colocalizes in endosomes and interacts with Wls. Wls becomes unstable in the absence of retromer activity. Our findings link Wls and retromer functions in the same conserved Wnt secretion pathway. We propose that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN).     

Wingless secretion promotes and requires retromer-dependent cycling of Wntless

Wnt ligands are lipid-modified, secreted glycoproteins that control multiple steps during embryogenesis and adult-tissue homeostasis. Little is known about the mechanisms underlying Wnt secretion. Recently, Wntless (Wls/Evi/Srt) was identified as a conserved multi-pass transmembrane protein whose function seems to be dedicated to promoting the release of Wnts. Here, we describe Wls accumulation in the Golgi apparatus of Wnt/Wingless (Wg)-producing cells in Drosophila, and show that this localization is essential for Wg secretion. Moreover, Wls localization and levels critically depend on retromer, a conserved protein complex that mediates endosome-to-Golgi protein trafficking in yeast. In the absence of the retromer components Dvps35 or Dvps26, but in presence of Wg, Wls is degraded and Wg secretion impaired. Our results indicate that Wg, clathrin-mediated endocytosis and retromer sustain a Wls traffic loop from the Golgi to the plasma membrane and back to the Golgi, thereby enabling Wls to direct Wnt secretion.     

Differential palmit(e)oylation of Wnt1 on C93 and S224 residues has overlapping and distinct consequences

Though the mechanisms by which cytosolic/intracellular proteins are regulated by the post-translational addition of palmitate adducts is well understood, little is known about how this lipid modification affects secreted ligands, such as Wnts. Here we use mutational analysis to show that differential modification of the two known palmit(e)oylated residues of Wnt1, C93 and S224, has both overlapping and distinct consequences. Though the relative roles of each residue are similar with respect to stability and secretion, two distinct biological assays in L cells show that modification of C93 primarily modulates signaling via a ß-catenin independent pathway while S224 is crucial for ß-catenin dependent signaling. In addition, pharmacological inhibition of Porcupine (Porcn), an upstream regulator of Wnt, by IWP1, specifically inhibited ß-catenin dependent signaling. Consistent with these observations, mapping of amino acids in peptide domains containing C93 and S224 demonstrate that acylation of C93 is likely to be Porcn-independent while that of S224 is Porcn-dependent. Cumulatively, our data strongly suggest that C93 and S224 are modified by distinct enzymes and that the differential modification of these sites has the potential to influence Wnt signaling pathway choice.     

Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.     

Sailing with the Wnt: Charting the Wnt processing and secretion route

Wnt proteins are members of a highly conserved family of signalling molecules that play a central role in development and disease. During the past years, the different signalling pathways that are triggered by Wnt proteins have been studied in detail, but it is still largely unknown how a functional Wnt protein is produced and secreted. The recent finding that Wnt proteins are post-translationally modified and the discovery of the Wnt binding protein Wntless and its trafficking by the retromer complex show that Wnt secretion is a complex and highly regulated process. In this review, we will give an overview of the Wnt maturation and secretion pathway and discuss how this process may influence the spreading and signalling activity of Wnt.     

Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, we demonstrated that this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process.     

ERAD‐dependent control of the Wnt secretory factor Evi

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.     

Wnts need a p(assport)24 to leave the ER

Wnts are secreted through a dedicated exocytic pathway, which has been only partly characterized. Here, Palmer and colleagues comment on two recent reports by the groups of K. Basler and M. Boutros, respectively, in which they show that p24 proteins take part in this exocytic route and are required for Wnt exit from the ER to the Golgi.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.212Wnt signalling proteins regulate diverse cellular processes during development and homeostasis. Since misregulation of Wnt signalling is associated with cancer, most research has been aimed at characterizing the signal transduction pathway. Recently, attention has focused on Wnt production due to the identification of factors required specifically for Wnt secretion. For instance, the specific requirement of Evi, also known as Wntless or Sprinter, suggested that Wnts might follow a specialized secretory route (Banziger et al, 2006; Bartscherer et al, 2006). Two recent papers published in EMBO reports by the groups of Konrad Basler in last month''s issue, and Michael Boutros in this issue, show that Wnt secretion requires the activity of p24 family members (Buechling et al, 2011; Port et al, 2011). Therefore, the specialized route might begin at endoplasmic reticulum (ER) exit sites.Wnts are secreted glycoproteins that can act many cell diameters from their source of production. Most, but not all Wnt proteins, are acylated and thus associate tightly with cellular membranes. Despite this association, acylated Wnts can be released from secreting cells and spread in the extracellular space (Bartscherer & Boutros, 2008; Port & Basler, 2010). Acylation of Wnts, which occurs in the ER, is thought to be mediated by the N-acetyl transferase encoded by porcupine (porc; van den Heuvel et al, 1993). After acylation, Wnt proteins associate with Evi, a multipass transmembrane protein found mostly at the Golgi and the plasma membrane (Fig 1A). This association is essential for the secretion of the Wnts that are acylated since, in the absence of Evi, they accumulate on internal membranes (Banziger et al, 2006; Bartscherer et al, 2006). It is therefore thought that acylated Wnts require Evi to exit the Golgi and progress to the cell surface (Port et al, 2008). This does not seem to be a requirement for non-lipidated Wnts, such as Drosophila WntD, which are secreted in the absence of Evi (Ching et al, 2008). Similarly, secretion of other signalling proteins proceeds normally without Evi. After reaching the cell surface, Evi is likely to have a choice between various routes. One such route, which involves the retromer complex, takes it back to the Golgi where it can participate in another round of Wnt secretion (Fig 1A). Alternatively, Evi can be targeted to lysosomes (Bartscherer & Boutros, 2008; Port & Basler, 2010). The factors that determine Evi transport remain poorly understood. Nevertheless, these studies highlight the essential and specific role of Evi for the secretion of lipidated Wnts.Open in a separate windowFigure 1Model summarizing the suggested roles of p24 proteins in endoplasmic reticulum to Golgi transport of Wg. (A) Schematic of a cell showing the current model of the Wg secretory pathway. Wg is produced in the ER where it is lipid-modified by Porc, and moved to the Golgi with the assistance of p24 proteins. In the Golgi, Wg joins Evi, which facilitates Wg transport to the cell surface. Evi is then recycled back to the Golgi in a path mediated by the retromer complex. (B)(i) In the absence of p24 proteins, there is no recruitment of Wg to COPII-coated vesicles and therefore a block of secretion in the ER. (ii) Port et al (2011) propose a similar model in which CHOp24/Emp24 and Éclair are involved in Wg recruitment, although only CHOp24/Emp24 binds to Wg. (iii) According to Buechling et al (2011), Opm recruits Wg to COPII-coated vesicles for movement to the Golgi. CHOp24 and p24-1 are also required for this process. ER, endoplasmic reticulum.Two groups have now reported the use of cell-based RNA interference (RNAi) screens to identify further proteins required for the secretion of Wingless (Wg), the main Drosophila Wnt (Buechling et al, 2011; Port et al, 2011). They found that p24 family members, a group of proteins previously implicated in both retrograde and anterograde transport between the ER and Golgi (Strating & Martens, 2009), are required for Wg secretion by S2 cells. Similarly, knockdown by transgenic RNAi shows that p24 proteins are required for normal levels of Wg secretion in Drosophila wing imaginal discs (Buechling et al, 2011; Port et al, 2011). As with Evi, this requirement seems to be relatively specific, since general secretion and the secretion of other signalling proteins, including the lipid-modified morphogen Hedgehog, are unaffected by p24 knockdown. Buechling et al also assessed the role of p24 proteins in WntD secretion. They found that RNAi against opossum (opm), one of the p24 members, prevents WntD secretion in cultured cells. They also show that the phenotypes of opm mutants and WntD mutant embryos resemble each other (Buechling et al, 2011). Therefore, while Evi is specifically required for the secretion of acylated Wnts, p24 proteins could contribute to the secretion of all Wnts. This function is likely to be conserved since the mammalian homologue of Opm, TMED5, is required for Wnt1 signalling, at least in a mammalian cell culture assay (Buechling et al, 2011).To gain understanding of the role of p24 proteins in Wnt secretion, both groups analysed the subcellular localization of Wg following p24 knockdown. They found accumulation in the ER and concomitant depletion in the Golgi, as indicated by reduced co-localization with Golgi markers (Fig 1Bi). They also found that p24 knockdown prevents Wg from stabilizing Evi in producing cells, suggesting that the stabilizing influence of Wg requires its exit from the ER (Buechling et al, 2011; Port et al, 2011). These results lead the authors to propose that the loss of p24 prevents the transport of Wg from the ER to the Golgi. Importantly, immunoprecipitation experiments suggest that Wg might interact physically with Opm and Emp24 (also known as CHOp24). This led both sets of authors to postulate a model whereby p24 proteins act as cargo receptors to escort Wnt proteins from the ER to the Golgi, whereupon they can bind to Evi, which will escort them to the plasma membrane. Thus, in this context, p24 proteins seem to have an anterograde function.Although both studies highlight the role of p24 proteins in Wnt secretion, they disagree on the relative importance of the various family members. Among the nine predicted p24 proteins encoded by the Drosophila genome, only Éclair and Emp24/CHOp24 were found to be required for Wg secretion by Port et al (2011; Fig 1Bii). By contrast, Buechling et al found that Opm, Emp24/CHOp24 and p24-1 all play a role in Wg secretion (fig 1Biii). Thus, only Emp24/CHOp24 is found by both groups to be essential for Wg secretion. Although functional redundancy among p24 proteins could explain why the removal of a single p24 protein has a relatively weak phenotype, there is no simple explanation as to why the very similar assays used by the two groups do not lead to identical conclusions. These differences could be worked out by the exchange of reagents and protocols.Regardless of the discrepancies, the two studies provide an important step in our understanding of Wnt secretion by demonstrating that Wnts engage with specialized components of the secretory machinery as early as in the ER. It might be relevant that the anterograde function of p24 proteins is directed at glycophosphatidylinositol (GPI)-anchored proteins, which have been shown to partition in raft-like microdomains (Strating & Martens, 2009). It is conceivable that GPI-anchored proteins, as well as Wnts, gather in a subdomain of the ER where they could both interact with p24 proteins and set off along their specialized secretory pathways. Wnt targeting to specialized membrane domains could in principle be mediated by their lipid moieties (Bartscherer & Boutros, 2008; Port & Basler, 2010). However, the process might turn out to be more complex if it is confirmed that p24 proteins are also required for the secretion of non-acylated Wnts (for example, WntD), as suggested by Buechling et al. In any case, it will be interesting to determine the precise molecular mechanism underlying the functional interaction between Wnts and p24 proteins as it is likely to explain how Wnts are allowed to exit the ER and start their journey out of the cell.     

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.     

Lipid Modification of Secreted Signaling Proteins

Proteins of the Hedgehog, Wnt and Epidermal Growth Factor Receptor (EGFR) ligand families are secreted signals that induce concentration-dependent responses in surrounding cells. Although these proteins must diffuse through the aqueous extracellular environment, recent work has shown that hydrophobic lipid modifications are essential for their functions. All three classes of ligands are palmitoylated in the secretory pathway by related enzymes, and Hedgehog also carries a C-terminal cholesterol modification as a result of its autocatalytic cleavage. Palmitoylation is required for Wingless secretion and contributes to the signaling activity of Hedgehog and Wnt3a, but is not required for secretion or receptor activation by the EGFR ligand Spitz. While lipid modifications enhance the long-range activity of Sonic hedgehog, they restrict the range and increase the local concentration of Spitz. We discuss the diverse functions and the possible extent of palmitoylation of secreted ligands.     

Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.