Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH₂ to produce PGD₂. Three cysteine residues, Cys⁶⁵, Cys⁸⁹, and Cys¹⁸⁶ in L-PGDS, are conserved among all species, of which Cys⁸⁹ and Cys¹⁸⁶ residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Proteolysis of SNAP-25 Isoforms by Botulinum Neurotoxin Types A, C, and E

Abstract : Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg¹⁹⁸ and Ala¹⁹⁹, depends on the presence of regions Asn⁹³ to Glu¹⁴⁵ and Ile¹⁵⁶ to Met²⁰², and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex ( K _D = 1.9 × 10^-7 M ) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met¹⁴⁶-Gln¹⁹⁷, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile¹⁵⁶-Asp¹⁸⁶. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys¹⁸⁵Asp or Pro¹⁸²Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.     

SNAP-25 and synaptotagmin 1 function in Ca2+-dependent reversible docking of granules to the plasma membrane

In neuroendocrine cells, Ca²⁺ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca²⁺ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT-20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca²⁺ concentrations in the micromolar range, is reversible and requires intact SNAP-25, but not VAMP-2. In the docking assay, addition of Ca²⁺ induces the formation of a SNAP-25-Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti-Synaptotagmin 1 antibodies block granule docking. These results show that Ca²⁺ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca²⁺-dependent interaction between SNAP-25 and Synaptotagmin 1 .     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

SNAP-23 and SNAP-25 are palmitoylated in vivo.

The neuronal presynaptic membrane t-SNARE complex consists of the transmembrane protein syntaxin with the palmitoylated protein SNAP-25. In non-neuronal tissues, SNAP-23 replaces SNAP-25 in the t-SNARE complex, although the mechanism of membrane anchoring of SNAP-23 has not been determined. We now report that like SNAP-25, SNAP-23 is palmitoylated in vivo on one or more cysteine residues present in a central "palmitoylation domain." Interestingly, SNAP-23 is palmitoylated less well than SNAP-25, and in vivo binding studies indicate a correlation between the extent of palmitoylation and the ability of SNAP-23 or SNAP-25 to bind to syntaxin in vivo.     

Role of the cysteine-rich domain of the t-SNARE component, SYNDET, in membrane binding and subcellular localization

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.     

Dopamine Transporter Cysteine Mutants: Second Extracellular Loop Cysteines Are Required for Transporter Expression

Abstract: Studies with thiol-modifying reagents have suggested that cysteines might play important roles in the function of the dopamine transporter (DAT). To identify DAT cysteines with important thiol groups, we have studied six mutant dopamine transporters in which cysteines were replaced by alanines. Substitutions of cysteines assigned to the DAT's second putative extracellular loop—positions 180 and 189—dramatically decreased the expression of the mutant transporters. Substitutions at positions 90, 242, 305, and 345 had no significant effect in decreasing dopamine uptake, MPP⁺ uptake, or cocaine analogue binding. Immunostaining COS cells transfected with Cys¹⁸⁰ and Cys¹⁸⁹ to Ala mutants revealed reduced membrane staining and prominent staining in perinuclear regions consistent with Golgi apparatus. These results suggest that cysteines in the DAT second extracellular loop may provide sulfide residues crucial to full transporter expression, at least in part, through interference with membrane insertion. Conceivably, they might also provide the targets for the influences of thiol-modifying reagents in modifying the function of the wild-type DAT expressed in striatal membranes.     

SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.     

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys⁻ mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR3_1–145 from multidrug resistance protein into SNAP-23 Cys⁻ mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys⁻ and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys⁻-MDR3_1–145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.     

Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin

The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.     

Palmitoylation of carboxypeptidase D. Implications for intracellular trafficking

Covalent lipid modifications mediate protein-membrane and protein-protein interactions and are often essential for function. The purposes of this study were to examine the Cys residues of the transmembrane domain of metallocarboxypeptidase D (CPD) that could be a target for palmitoylation and to clarify the function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the plasma membrane. We constructed AtT-20 cells stably expressing various constructs carrying a reporter protein (albumin) fused to a transmembrane domain and the CPD cytoplasmic tail. Some of the constructs contained the three Cys residues present in the CPD transmembrane region, while other constructs contained Ala in place of the Cys. Constructs carrying Cys residues were palmitoylated, while those constructs lacking the Cys residues were not. Because palmitoylation of several proteins affects their association with cholesterol and sphingolipid-rich membrane domains or caveolae, we tested endogenous CPD and several of the reporter constructs for resistance to extraction with Triton X-100. A construct containing the Cys residues of the CPD transmembrane domain was soluble in Triton X-100 as was endogenous palmitoylated CPD, indicating that palmitoylation does not target CPD to detergent-resistant membrane rafts. Interestingly, constructs of CPD that lack palmitoylation sites have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6- phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting

We have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS- PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of approximately 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys34 to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys30 to Ala mutation had no biologic consequences, showing the importance of Cys34. Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys30 and Cys34 and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys30 and Cys34 leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys34 is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.     

The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Regulation by distinct cysteine-rich domains

SNAP-25 and its ubiquitously expressed homologue, SNAP-23, are SNARE proteins that are essential for regulated exocytosis in diverse cell types. Recent work has shown that SNAP-25 and SNAP-23 are partly localized in sphingolipid/cholesterol-rich lipid raft domains of the plasma membrane and that the integrity of these domains is important for exocytosis. Here, we show that raft localization is mediated by a 36-amino-acid region of SNAP-25 that is also the minimal sequence required for membrane targeting; this domain contains 4 closely spaced cysteine residues that are sites for palmitoylation. Analysis of endogenous levels of SNAP-25 and SNAP-23 present in lipid rafts in PC12 cells revealed that SNAP-23 (54% raft-associated) was almost 3-fold more enriched in rafts when compared with SNAP-25 (20% raft-associated). We report that the increased raft association of SNAP-23 occurs due to the substitution of a highly conserved phenylalanine residue present in SNAP-25 with a cysteine residue. Intriguingly, although the extra cysteine in SNAP-23 enhances its raft association, the phenylalanine at the same position in SNAP-25 acts to repress the raft association of this protein. These different raft-targeting signals within SNAP-25 and SNAP-23 are likely important for fine-tuning the exocytic pathways in which these proteins operate.     

Melatonin Biosynthesis in Cultured Chick Retinal Photoreceptor Cells: Calcium and Cyclic AMP Protect Serotonin N-Acetyltransferase from Inactivation in Cycloheximide-Treated Cells

Abstract: The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N -acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 µ M forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of ∼30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K⁺, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K⁺ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca²⁺ influx through dihydropyridine-sensitive channels. MDL-12330A, an inhibitor of K⁺-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K⁺. This result suggests that the effect of Ca²⁺ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization-evoked Ca²⁺ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.     

SNAP-25 is targeted to the plasma membrane through a novel membrane-binding domain.

SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membrane-targeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two alpha-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: P₀ glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P₀ was labeled in rat sciatic nerve slices with [³H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M NaBH₄ with the concomitant production of [³H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [¹⁴C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [¹⁴C]carboxyamidomethylated P₀was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys¹⁵³ in rat P₀ glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.