RESUMO
Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Leveduras/química , Sequência Conservada , Análise Mutacional de DNA , Deleção de Genes , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Qa-SNARE , Proteínas SNARE , Relação Estrutura-AtividadeRESUMO
ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.
Assuntos
AMP Cíclico/metabolismo , Família Multigênica , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Sequência Conservada/genética , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Cricetinae , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Especificidade de Órgãos , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/genética , Fosforilação , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.
Assuntos
Proteínas de Transporte , Sequência Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Proteínas Fúngicas/genética , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Qa-SNARE , Saccharomyces cerevisiae/química , Alinhamento de Sequência , Relação Estrutura-Atividade , Sintaxina 1RESUMO
Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.
Assuntos
Dopamina/farmacologia , Neostriado/enzimologia , Neurônios/enzimologia , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células COS , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática , Feminino , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/genética , TransfecçãoRESUMO
Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.
Assuntos
Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Exocitose , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/genética , Sítios de Ligação , Células COS , Humanos , Proteínas Munc18 , Mutagênese , Proteínas do Tecido Nervoso/genética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Células PC12 , Conformação Proteica , Prótons , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Sintaxina 1RESUMO
Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.
Assuntos
Antígenos de Superfície/química , Antígenos de Superfície/genética , Evolução Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Animais , Antígenos de Superfície/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Espectroscopia de Ressonância Magnética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica/fisiologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Transmissão Sináptica/fisiologia , Sinaptotagmina I , Sinaptotagminas , Sintaxina 1RESUMO
The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+, K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+, K+-ATPase alpha1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha1 and suggest a connection between this enzyme and cell adhesion.
Assuntos
Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Animais , Células COS , Adesão Celular , Tamanho Celular , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Ratos , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.
Assuntos
Proteína Quinase C/metabolismo , Serina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Inibidores Enzimáticos , Amarelo de Eosina-(YS) , Corantes Fluorescentes , Sondas Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fosfopeptídeos/análise , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
Assuntos
Viúva Negra/metabolismo , Expressão Gênica , Venenos de Aranha/biossíntese , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Anquirinas/química , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Primers do DNA , DNA Complementar , Escherichia coli , Insetos , Bicamadas Lipídicas , Espectrometria de Massas , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Peso Molecular , Músculos/efeitos dos fármacos , Músculos/fisiologia , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Venenos de Aranha/farmacologiaRESUMO
The structural gene of delta-latroinsectotoxin was cloned and its nucleotide sequence was determined. The gene contains an open reading frame of 3642 bp. The deduced amino acid sequence is homologous to the sequences of latrotoxins studied earlier.
Assuntos
Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Viúva Negra , Clonagem Molecular , Dados de Sequência Molecular , Fases de Leitura AbertaRESUMO
alpha-Latroinsectotoxin (alpha-LIT), purified from venom glands of the black widow spider Latrodectus mactans tredecimguttatus, is a presynaptic neurotoxin selective only for insects. A cDNA encoding the putative alpha-LIT precursor was isolated from a spider venom gland cDNA library. The cDNA contains a 4236-base-pair open reading frame corresponding to a 157826-Da protein composed of 1411 amino acids. The mature alpha-LIT, with molecular mass approximately 130 kDa, is probably derived from double processing in the N-terminal and C-terminal regions of the primary translation product. The structure region, extending over residues 464-1176, is composed almost entirely of ankyrin-like repeats which represent a motif also found in the alpha-latrotoxin (alpha-LTX), which has selective action on vertebrates. Total alignment of the alpha-LIT and alpha-LTX amino acid sequences reveals an overall similarity of 34.1%. Strong sequence divergence is observed in analogous cysteine-rich regions situated within the ankyrin-repeat domains of both alpha-LIT and alpha-LTX.
Assuntos
Anquirinas/genética , Sequências Repetitivas de Ácido Nucleico , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Viúva Negra , Clonagem Molecular , DNA , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Some samples of latrotoxin purified from the black widow spider venom contain two components: alpha-latrotoxin (M(r) approximately 130,000) and a low mol. wt protein with M(r) about 8000. Clones carrying the cDNA sequence for the low mol. wt protein copurified with alpha-latrotoxin were isolated from spider venom glands. Nucleotide sequence analysis of the cloned cDNA revealed the primary structure of the polypeptide to be 18 amino acids signal peptide and 70 amino acids protein chain with mol. wt of 7947 and pI of approximately 4.0. The protein exhibits certain structural homology with erabutoxin-a from the sea snake.
Assuntos
Proteínas/isolamento & purificação , Venenos de Aranha/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Peso MolecularRESUMO
cDNA encoding the putative alpha-latrotoxin precursor was isolated from spider venom glands cDNA library and sequenced. The cDNA contained the 4203 base-pair open reading frame corresponding to the 156,855-Da protein composed of 1401 amino acids. Computer analysis of the deduced primary structure revealed the presence of various internal imperfect repeats mainly in its central and C-terminal regions.
Assuntos
Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Biblioteca Gênica , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Poli A/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Venenos de Aranha/químicaRESUMO
The primary structure of a gene of the Na+, K+-ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical alpha III-form of the enzyme catalytic subunit. The gene comprises over 25,000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Bacteriófago lambda/genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Catálise , DNA/genética , DNA Recombinante , Éxons , Humanos , Íntrons , Dados de Sequência MolecularAssuntos
ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Células HeLa/enzimologia , Humanos , Rim/enzimologia , Substâncias Macromoleculares , Hibridização de Ácido Nucleico , Ratos , Ovinos , SuínosRESUMO
The neurotoxin Os-1 from the venom of the Central Asian scorpion Orthochirus scrobiculosus possesses a high paralytic activity against mice. This neurotoxin was subjected to tryptic, chymotryptic and BrCN cleavages and its total amino acid sequence was established. It was shown that neurotoxin Os-1 consists of 66 amino acid residues an contains four disulfide bonds.
Assuntos
Neurotoxinas/análise , Venenos de Escorpião/análise , Sequência de Aminoácidos , Animais , Brometo de Cianogênio , Hidrólise , Neurotoxinas/isolamento & purificação , Oligopeptídeos/análise , TripsinaRESUMO
Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.