Release of fatty acids from virus glycoproteins by hydroxylamine

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.     

Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus.

The cysteine residue in the cytoplasmic domain at position 489 of the sequence of the glycoprotein (G protein) isolated from vesicular-stomatitis virions is completely blocked for carboxymethylation. After release of covalently bound fatty acids by hydroxylamine at pH 6.8, this cysteine residue could be specifically labelled by iodo[14C]acetic acid. Reaction products were analysed after specific cleavage of labelled G protein at asparagine-glycine bonds by hydroxylamine at pH 9.3, which generated a C-terminal peptide of Mr 15,300 containing only the single cysteine residue. Bromelain digestion of [3H]palmitic acid-labelled membrane fractions of vesicular-stomatitis-virus-infected baby-hamster kidney cells removed almost completely the 3H radioactivity from the cytoplasmic domain of the G protein, whereas the ectodomain was completely protected by the microsomal membrane. This result indicates that the acylation site of the G protein is exposed on the cytoplasmic side of intracellular membranes. Taken together, both biochemical techniques strongly suggest that the single cysteine-489 residue, which is located six amino acid residues distal to the putative transmembrane domain, is the acylation site. The thioester bond between palmitic acid and the G protein is quite resistant to hydroxylamine treatment (0.32 M at pH 6.8 for 1 h at 37 degrees C) compared with the reactivity of the thioester linkage in palmitoyl-CoA, which is cleaved at relatively low concentrations of hydroxylamine (0.05 M).     

Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein

Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

SNARE proteins catalyze many forms of biological membrane fusion, including Ca²⁺-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca²⁺-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca²⁺-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals.     

Synapsin I-mediated interaction of brain spectrin with synaptic vesicles

We have established a new binding assay in which 125I-labeled synaptic vesicles are incubated with brain spectrin covalently immobilized on cellulosic membranes in a microfiltration apparatus. We obtained saturable, high affinity, salt- (optimum at 50-70 mM NaCl) and pH- (optimum at pH 7.5-7.8) dependent binding. Nonlinear regression analysis of the binding isotherm indicated one site binding with a Kd = 59 micrograms/ml and a maximal binding capacity = 1.9 micrograms vesicle protein per microgram spectrin. The fact that the binding of spectrin was via synapsin was demonstrated in three ways. (a) Binding of synaptic vesicles to immobilized spectrin was eliminated by prior extraction with 1 M KCl. When the peripheral membrane proteins in the 1 M KCl extract were separated by SDS-PAGE, transferred to nitrocellulose paper and incubated with 125I-brain spectrin, 96% of the total radioactivity was associated with five polypeptides of 80, 75, 69, 64, and 40 kD. All five polypeptides reacted with an anti-synapsin I polyclonal antibody, and the 80- and 75-kD polypeptides comigrated with authentic synapsin Ia and synapsin Ib. The 69- and 64-kD polypeptides are either proteolytic fragments of synapsin I or represent synapsin IIa and synapsin IIb. (b) Pure synapsin I was capable of competitively inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin with a Kl = 46 nM. (c) Fab fragments of anti-synapsin I were capable of inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin. These three observations clearly establish that synapsin I is a primary receptor for brain spectrin on the cytoplasmic surface of the synaptic vesicle membrane.     

Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.     

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.     

极性蛋白Crumbs胞内域功能保守性研究

跨膜蛋白Crumbs(Crb)是细胞顶部的决定因子,对上皮细胞顶-底极性的建立和维持起着关键的作用。其胞内域虽然仅有37个氨基酸,但对Crb的功能必不可少。在果蝇(Drosophila melanogaster)中,如果胞内域发生突变,将造成胚胎发育异常、上皮细胞顶底极性丧失等严重后果。Crb胞内域从果蝇到小鼠(Mus musculus)和人类(Homo sapiens)具有很高的同源性,但线虫(Caenorhabditis elegans)两个Crb蛋白的胞内域与果蝇和哺乳动物却较为不同。为验证线虫Crb蛋白胞内域是否功能保守,本文利用基因组工程法(Genomic engineering),将果蝇基因组中Crb基因编码胞内域的部分替换为一致性和相似性较远的线虫Crb2基因的相应区段。与其他Crb胞内域突变果蝇不同,替换突变体胚胎发育正常,Crb及其他极性蛋白的表达和定位正常,胚胎上皮细胞顶底极性能够正确的建立和维持。这些结果证实虽然线虫和果蝇Crb蛋白胞内域之间存在大量序列变异,但重要的氨基酸位点和功能模块则完全保守。     

Fatty acylation of human platelet proteins: evidence for myristoylation of a 50 kDa peptide

Fatty acid acylation of platelet proteins was studied by measuring incorporation of [3H]palmitate and [3H]myristate after incubation at 37 degrees C for 4 h. About ten major radiolabeled proteins were detected after SDS-polyacrylamide gel electrophoresis and fluorography, for both fatty acids. Cleavage by hydroxylamine treatment indicated an ester bond of either palmitate or myristate to these proteins. Nevertheless, a single 50 kDa peptide was specifically modified by an amide-linked myristate. The functions of acylated proteins in platelets are still unknown, but their relation with DLPC-induced shape changes and vesicle shedding is excluded.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with [3H]palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.     

Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters.

The p-nitrophenyl esters of straight-chain fatty acids were used as substrates of the enzyme subtilisin Novo (EC 3.4.4.16) and its chemically produced artificial enzyme thiolsubtilisin. Subtilisin and thiolsubtilisin pH--activity profiles were determined, and kinetic effects of the active site O-S substitution were observed. Among the substrates tested, both enzymes show highest specificity with p-nitrophenyl butyrate. It was also found that subtilisin is more sensitive to changes in substrate chain length than is thiolsubtilisin. Second-order acylation rate constants (k2/Ks) are remarkably similar for both enzymes. However, thiolsubtilisin deacylation rate constants and Km values are lower than analogous subtilisin constants. While thiolsubtilisin deacylation rate constants give a pH profile identical with that of subtilisin, the pH profile of thiolsubtilisin acylation rate constants shows an active site pK value lowered from the subtilisin pK of 7.15 and exhibits an inflection point at pH 8.45, which is absent in subtilisin.     

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.     

Changes in mucus glycoprotein synthesized in rat gastric mucosa exposed to ethanol

The resistance to proteolysis by pepsin of gastric mucus glycoprotein synthesized by tissue culture in the presence and absence of 0.1 M ethanol was investigated. The glycoprotein product of ethanol-supplemented culture was found to contain 68% less associated lipids and 81% less covalently bound fatty acids, but exhibited unaltered content of carbohydrate and protein. The lipid and fatty acyl deficient glycoprotein was 5-times more rapidly and 2-3-times more extensively degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Following delipidation with organic solvents and deacylation with hydroxylamine both glycoproteins were digested at the same rate and degraded to the same extent. The lower content of fatty acyl residues markedly affected the overall pattern of the proteolytic fragments identified by SDS gel electrophoresis. The peptides corresponding to the acylated fragments of control were degraded and an increase in the amount of smaller peptides was observed. The in vitro assays of the fatty acyltransferase activity towards the substrates obtained from control and alcohol-containing cultures revealed that the enzyme activity was similar and increased proportionally with increased concentration of both glycoprotein substrates and enzyme. However, addition of 0.1 M ethanol to the assay tubes containing complete incubation mixture decreased the acylation of either glycoprotein by 40%. Based on the results presented here, and on previous studies of mucus glycoprotein synthesis in the presence of ethanol, we conclude that ethanol interferes with the process of acylation of mucus glycoprotein with fatty acids.     

A trimeric protein complex functions as a synaptic chaperone machine

We identify a chaperone complex composed of (1) the synaptic vesicle cysteine string protein (CSP), thought to function in neurotransmitter release, (2) the ubiquitous heat-shock protein cognate Hsc70, and (3) the SGT protein containing three tandem tetratricopeptide repeats. These three proteins interact with each other to form a stable trimeric complex that is located on the synaptic vesicle surface, and is disrupted in CSP knockout mice. The CSP/SGT/Hsc70 complex functions as an ATP-dependent chaperone that reactivates a denatured substrate. SGT overexpression in cultured neurons inhibits neurotransmitter release, suggesting that the CSP/SGT/Hsc70 complex is important for maintenance of a normal synapse. Taken together, our results identify a novel trimeric complex that functions as a synapse-specific chaperone machine.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.