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1.
The addition of palmitate to cysteine residues enhances the hydrophobicity of proteins, and consequently their membrane association. Here we have investigated whether this type of fatty acylation also regulates protein-protein interactions. GAP-43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N-terminus of GAP-43 are subject to palmitoylation, and are necessary for membrane binding as well as for G(o) activation. N-terminal peptides, which include these cysteines, stimulate G(o). Monopalmitoylation reduces, and dipalmitoylation abolishes the activity of the peptides. The activity of GAP-43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP-43 between an acylated, membrane-bound reservoir of inactive GAP-43, and a depalmitoylated, active pool of protein.  相似文献   

2.
The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.  相似文献   

3.
S G Coats  M A Booden  J E Buss 《Biochemistry》1999,38(39):12926-12934
H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.  相似文献   

4.
Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.  相似文献   

5.
Schey KL  Gutierrez DB  Wang Z  Wei J  Grey AC 《Biochemistry》2010,49(45):9858-9865
Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.  相似文献   

6.
Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.  相似文献   

7.
To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [3H]myristic acid or [3H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.  相似文献   

8.
Palmitoylcarnitine was previously shown to promote differentiation of neuroblastoma NB-2a cells. It was also observed to increase palmitoylation of several proteins and to diminish incorporation of palmitic acid to phospholipids, as well as to affect growth associated protein GAP-43 by decreasing its phosphorylation and interaction with protein kinase C. The present study was focused on influence of palmitoylcarnitine on palmitoylation of GAP-43 and lipid metabolism. Althought palmitoylcarnitine did not significantly affect the total phospholipids and fatty acid content, it increased incorporation of palmitate moiety to triacylglicerides and cholesterol esters, with a decrease of free cholesterol content. The presence of palmitoylcarnitine significantly increased the amount of covalently bound palmitate to GAP-43, which can regulate the signal transduction pathways.  相似文献   

9.
Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16-carbon fatty acid palmitate, is the most common acylation of proteins in eukaryotic cells. This post-translational modification provides an important mechanism for regulating protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, interaction with effectors and other aspects of protein function. In addition, N-terminal myristoylation and C-terminal prenylation, two well-studied post-translational modifications, frequently precede protein S-palmitoylation at a nearby spot of the polypeptide chain. Whereas N-myristoylation and prenylation are considered essentially irreversible attachments, S-palmitoylation is a tightly regulated, reversible modification. In addition, the unique reversibility of protein palmitoylation also allows proteins to rapidly shuttle between intracellular membrane compartments in a process controlled, in some cases, by the DHHC family of palmitoyl transferases. Recent cotransfection experiments using the DHHC family of protein palmitoyl transferases as well as RNA interference results have revealed that these enzymes, frequently localized to the Golgi apparatus, tightly control subcellular trafficking of acylated proteins. In this article we will give an overview of how protein palmitoylation regulates protein trafficking and subcellular localization.  相似文献   

10.
Neuronal protein GAP-43 performs multiple functions in axon guidance, synaptic plasticity and regulation of neuronal death and survival. However, the molecular mechanisms of its action in these processes are poorly understood. We have shown that in axon terminals GAP-43 is a substrate for calcium-activated cysteine protease m-calpain, which participates in repulsion of axonal growth cones and induction of neuronal death. In pre-synaptic terminals in vivo, in synaptosomes, and in vitro, m-calpain cleaved GAP-43 in a small region near Ser41, on either side of this residue. In contrast, micro-calpain cleaved GAP-43 in vitro at several other sites, besides Ser41. Phosphorylation of Ser41 by protein kinase C or GAP-43 binding to calmodulin strongly suppressed GAP-43 proteolysis by m-calpain. A GAP-43 fragment, lacking about forty N-terminal residues (named GAP-43-3), was produced by m-calpain-mediated cleavage of GAP-43 and inhibited m-calpain, but not micro-calpain. This fragment prevented complete cleavage of intact GAP-43 by m-calpain as a negative feedback. GAP-43-3 also blocked m-calpain activity against casein, a model calpain substrate. This implies that GAP-43-3, which is present in axon terminals in high amount, can play important role in regulation of m-calpain activity in neurons. We suggest that GAP-43-3 and another (N-terminal) GAP-43 fragment produced by m-calpain participate in modulation of neuronal response to repulsive and apoptotic signals.  相似文献   

11.
Characterization of the Palmitoylation Domain of SNAP-25   总被引:5,自引:2,他引:3  
Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys85,88 or Cys90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.  相似文献   

12.
An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.  相似文献   

13.
An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.  相似文献   

14.
Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.  相似文献   

15.
The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.  相似文献   

16.
The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane- association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP- 43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin- containing cortical cytoskeleton in a regulatable manner.  相似文献   

17.
Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(DeltaN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(DeltaN168) when coexpressed in COS 7 cells and that palmitoylation of Cys(240) and Cys(241) was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys(223) and Cys(224) in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(DeltaN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.  相似文献   

18.
Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.  相似文献   

19.
Abstract: The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35–50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated Go but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser41 inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.  相似文献   

20.
Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.  相似文献   

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