Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro.

We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C.     

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.     

Annexin VI Binds to a Synaptic Vesicle Protein, Synapsin I

Abstract: Annexin VI bound to >14 species of proteins in the whole homogenate of rat forebrain in a Ca²⁺/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M_r 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca²⁺ ( K _Ca) was 12.6 µ M , and the affinity for annexin VI ( K _D) was ∼270 n M . Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca²⁺/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca²⁺- and phospholipid-dependent manner.     

Characterization of synapsin I binding to small synaptic vesicles

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.     

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.     

Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I.

A rat brain cDNA clone containing an open reading frame encoding the neuron-specific protein synapsin I has been sequenced. The sequence predicts a protein of 691 amino acids with a mol. wt of 73 kd. This is in excellent agreement with the size of rat brain synapsin Ib measured by SDS--polyacrylamide gel electrophoresis. Inspection of the predicted primary structure has revealed the probable sites for synapsin I phosphorylation by the cAMP-dependent and Ca2+/calmodulin-dependent protein kinases. All of the biochemically observed intermediates of synapsin I digestion by collagenase can be verified by inspection of the sequence, and the collagenase-resistant fragment has been defined as the amino-terminal 439 amino acids of the molecule. Predictions of sequence secondary structure and hydrophobicity suggest that a central domain of approximately 270 amino acids may exist as a folded, globular core. The carboxyl-terminal domain of the protein (the region sensitive to collagenase digestion) contains sites for Ca2+/calmodulin-dependent protein kinase phosphorylation. These sites are flanked by three regions of repeating amino acid sequence that are proposed to be the synaptic vesicle-binding domain of synapsin I. This region also shares homology with the actin-binding proteins profilin and villin. The characteristics of the synapsin I sequence do not support extensive homology with the erythrocyte cytoskeletal protein 4.1.     

Tetramerization and ATP binding by a protein comprising the A, B, and C domains of rat synapsin I

Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca(2+) and ATP reveal that this protein can form a tetramer and that a flexible loop (the "multifunctional loop") contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus.     

Homo- and heterodimerization of synapsins.

In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes. Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates. The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (>300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP. We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers. This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization. These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences. The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice.     

Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton and is believed to be involved in the modulation of neurotransmission. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.     

Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis.

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.     

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleocytoplasmic shuttling of dysbindin-1, a schizophrenia-related protein, regulates synapsin I expression

Dysbindin-1 is a 50-kDa coiled-coil-containing protein encoded by the gene DTNBP1 (dystrobrevin-binding protein 1), a candidate genetic factor for schizophrenia. Genetic variations in this gene confer a susceptibility to schizophrenia through a decreased expression of dysbindin-1. It was reported that dysbindin-1 regulates the expression of presynaptic proteins and the release of neurotransmitters. However, the precise functions of dysbindin-1 are largely unknown. Here, we show that dysbindin-1 is a novel nucleocytoplasmic shuttling protein and translocated to the nucleus upon treatment with leptomycin B, an inhibitor of exportin-1/CRM1-mediated nuclear export. Dysbindin-1 harbors a functional nuclear export signal necessary for its nuclear export, and the nucleocytoplasmic shuttling of dysbindin-1 affects its regulation of synapsin I expression. In brains of sandy mice, a dysbindin-1-null strain that displays abnormal behaviors related to schizophrenia, the protein and mRNA levels of synapsin I are decreased. These findings demonstrate that the nucleocytoplasmic shuttling of dysbindin-1 regulates synapsin I expression and thus may be involved in the pathogenesis of schizophrenia.     

Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins

Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.     

Binding of Protein Kinase Inhibitors to Synapsin I Inferred from Pair-Wise Binding Site Similarity Measurements

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-γ³⁵S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50  = 0.15 µM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90α inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Do synapsin I and microtubule-associated proteins bind to a common site on polymerized tubulin?

Synapsin I plays an important role in the regulation of neurotransmitter release, since it binds to synaptic vesicles and to the cytoskeleton, and it bundles F-actin and microtubules. We have previously shown by tryptic digestion of synapsin I that a 44 kDa fragment contains a binding site for polymerized tubulin. In the present experiments, we test whether synapsin I and microtubule-associated proteins (MAPs) have the same or a different binding site on tubulin molecules. Our results show that heat stable MAPs do not compete with synapsin I for binding to taxol tubulin. In addition, subtilisin digestion of tubulin, which suppresses MAPs binding, does not abolish synapsin I cosedimentation with taxol tubulin. Thus, our results strongly suggest that synapsin I (as reported for kinesin) does not bind to the 4 kDa subtilisin digested C-terminal part of the tubulin molecule.     

A Novel Synaptic Vesicle-Associated Phosphoprotein: SVAPP-120

Generation of antibodies and direct protein sequencing were used to identify and characterize proteins associated with highly purified synaptic vesicles from rat brain. A protein doublet of low abundance of 119 and 124 kDa apparent molecular mass [synaptic vesicle-associated phosphoprotein with a molecular mass of 120 kDa (SVAPP-120)] was identified using polyclonal antibodies. SVAPP-120 was found to copurify with synaptic vesicles and to be enriched in the purified synaptic vesicle fraction to the same extent as synapsin I. Like synapsin I, SVAPP-120 is not an integral membrane protein because it was released from synaptic vesicles by high salt concentrations. This protein was demonstrated to be brain specific, and its distribution in various brain regions paralleled the distribution of synapsin I and synaptophysin. During the postnatal development of the rat cortex and cerebellum, its expression correlated with synaptogenesis. SVAPP-120 was demonstrated to be a phosphoprotein both in vivo and in vitro. It was shown to be phosphorylated on serine and to a lesser extent on threonine residues. These results provide evidence that SVAPP-120 represents a novel synaptic vesicle-associated phosphoprotein. In addition, aldolase, a glycolytic enzyme, and alpha c-adaptin, a clathrin assembly-promoting protein, were identified on purified synaptic vesicles by direct protein sequencing.     

Synapsins Contain O-Linked N-Acetylglucosamine

The neuron-specific synaptic vesicle-associated phosphoproteins synapsin I and synapsin II were shown to contain terminal N-acetylglucosamine (GlcNAc) residues as determined by specific labeling with bovine galactosyltransferase and UDP-[3H]galactose. The beta-elimination of galactosyltransferase radiolabeled synapsin I and subsequent analysis of released saccharide on high-voltage paper electrophoresis confirmed the presence of monosaccharidic GlcNAc moieties in O-linkage to the protein. Partial cleavage of synapsin I by collagenase, 2-nitro-5-thiocyanobenzoic acid, and Staphylococcus aureus V8 protease suggests that at least three glycosylation sites exist along the molecule. Taken together these data present the first evidence that a neuron-specific protein contains O-glycosidically bound GlcNAc.     

Nectarin I is a novel,soluble germin-like protein expressed in the nectar of Nicotiana sp.

We have identified a limited number of proteins secreted into the nectar of tobacco plants. Nectarin I is the most highly expressed nectar protein and has a monomer molecular mass of 29 kDa. The other major nectar proteins are expressed at lower levels and have monomer molecular masses of 41, 54, and 65 kDa respectively. Nectarin I was purified and antiserum was raised against the protein. Under nondenaturing conditions, Nectarin I has an apparent molecular mass of >120 kDa. The expression of Nectarin I was restricted to nectary tissues and to a much lower level in the ovary. No Nectarin I was found in petals, stems, leaves, or roots or other floral tissues. The expression of Nectarin I was also developmentally regulated. It is expressed in nectary tissues only while nectar is being actively secreted. Subsequently, the N-terminus of purified Nectarin I was sequenced. Sequence identity showed Nectarin I is related to wheat germin. Although hydrogen peroxide is readily detectable in tobacco floral nectar, we were unable to demonstrate any oxalate oxidase activity for Nectarin I. A partial cDNA encoding the mature Nectarin I N-terminus was isolated and used to probe a Nicotiana plumbaginifolia genomic library. The Nectarin I gene was isolated and the translated sequence was consistent with both N-terminal and internal cyanogen bromide-derived amino acid sequence. The gene contains a single 386 nt intron and encodes a mature protein of 197 amino acids.     

Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein

The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general. A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii. The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E. coli. The 8.8 kDa gene product contains 81 amino acids. The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite. The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein. The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism.