RESUMO
α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa.
Assuntos
Actinina/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Actinina/genética , Proteínas Fúngicas/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Transporte Proteico , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismoRESUMO
Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.
Assuntos
Proteínas de Algas/análise , Proteínas Fúngicas/análise , Neurospora crassa/química , Phytophthora/química , Espectrina/análise , Proteínas de Algas/metabolismo , Animais , Especificidade de Anticorpos , Extratos Celulares/análise , Galinhas , Reações Cruzadas , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Soros Imunes/análise , Soros Imunes/imunologia , Neurospora crassa/metabolismo , Fator 2 de Elongação de Peptídeos/análise , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Phytophthora/metabolismo , Coelhos , Sensibilidade e Especificidade , Espectrina/imunologia , Espectrina/metabolismoRESUMO
An N(alpha)-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of M(r) 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl(2) and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the M(r) 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly N(alpha)-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discussed.
Assuntos
Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Quitridiomicetos/enzimologia , Acetilação , Sítios de Ligação , Antígenos CD13/química , Quelantes/farmacologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Isoflurofato/química , Isoflurofato/farmacologia , Cinética , Peso Molecular , Serina/química , Serina/metabolismo , Inibidores de Serina Proteinase/farmacologia , Frações Subcelulares/química , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Especificidade por Substrato , Temperatura , Zinco/farmacologiaRESUMO
Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.
Assuntos
Allomyces/enzimologia , Allomyces/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/fisiologia , Allomyces/crescimento & desenvolvimento , Allomyces/metabolismo , Sequência de Aminoácidos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Immunogold labeling of calcium-dependent neutral protease II (CDPII) with specific antibodies in near median longitudinal ultrathin sections of Allomyces arbuscula showed that the enzyme is predominantly localized in the growing hyphal and rhizoidal apices. The tips in both cell type had more enzyme than the distal regions and showed a gradient distribution. Labeling of the ultrathin sections and western blot analysis of purified subcellular fractions showed that CDPII is mainly cytosolic. Catalytic activity of the enzyme measured with synthetic substrate (Bz-Arg-pNA) showed that 90% of its activity is present in the soluble fraction, although a small amount is associated with the nuclei (0.2%), plasma membranes (0.7%) and microsomes (3.9%). This association is discussed in the context of the functional role of the enzyme and its possible localized activation. Western blot analysis of the crude extract and indirect immunofluorescence of the fixed permeabilized hypahe after treatment with CDPII showed that the alpha-tubulin is a specific target of the enzyme.