RESUMO
During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N-terminal extension may only play a role in C subunit degradation.
Assuntos
Blastocladiella/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endopeptidases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Expressão Gênica , Holoenzimas/metabolismo , Dados de Sequência Molecular , Recombinação GenéticaRESUMO
Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.
Assuntos
Blastocladiella/enzimologia , Dictyostelium/enzimologia , Neurospora crassa/enzimologia , Fosfotreonina/metabolismo , Animais , Especificidade por SubstratoRESUMO
In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.
Assuntos
Blastocladiella/enzimologia , Blastocladiella/genética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Blastocladiella/crescimento & desenvolvimento , Western Blotting , Clonagem Molecular , DNA Fúngico/análise , DNA Fúngico/genética , Membranas Intracelulares/enzimologia , Metaloendopeptidases/química , Dados de Sequência Molecular , Análise de Sequência de DNA , Partículas Submitocôndricas/enzimologia , Transcrição Gênica , Peptidase de Processamento MitocondrialRESUMO
Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism
Assuntos
Blastocladiella/enzimologia , Dictyostelium/enzimologia , Células Eucarióticas/enzimologia , Neurospora crassa/enzimologia , Fosfotreonina/metabolismo , Germinação/fisiologia , Especificidade por SubstratoRESUMO
A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.
Assuntos
Blastocladiella/enzimologia , Blastocladiella/genética , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Fúngicos , Biblioteca Genômica , Substâncias Macromoleculares , Metaloendopeptidases/química , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Transcrição Gênica , Peptidase de Processamento MitocondrialRESUMO
Chitin, a beta-(1-->4) polymer of N-acetyl-glucosamine, is an important constituent of fungal cell walls. This polymer is synthesized by the incorporation of N-acetyl-D-glucosamine units from the precursor UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) in a reaction catalyzed by chitin synthase. In the aquatic fungus Blastocladiella emersonii, chitin, the major component of the cell wall, is synthesized and incorporated in the cell surface of the free-swimming zoospore during the abrupt transition from this wall-less cell to the sessile, wall-containing cyst. Studies with cycloheximide indicate that chitin synthesis occurs in the apparent absence of protein synthesis, and thus posttranslational controls presumably regulate the cell wall biogenesis during encystment. Glutamine: fructose 6-phosphate amidotransferase, first enzyme of the hexosamine biosynthetic pathway, was found to play a central role in the regulation of chitin synthesis in this fungus. This enzyme exists in two forms, which are interconvertible by phosphorylation or dephosphorylation of serine residues. It is allosterically inhibited in the phosphorylated form, as it is in the zoospore, by UDP-GlcNAc. In addition, UDP-GlcNAc inhibits the dephosphorylation of amidotransferase catalyzed by protein phosphatases 2A and 2C. Thus, UDP-GlcNAc plays a dual role in hexosamine and chitin synthesis in zoospore: it not only inhibits the phosphorylated form of the enzyme but also prevents its dephosphorylation. The available data suggest that substrate availability plays a role in the control of chitin synthesis during zoospore differentiation.
Assuntos
Blastocladiella/fisiologia , Parede Celular/fisiologia , Hexosaminas/metabolismo , Blastocladiella/citologia , Blastocladiella/enzimologia , Quitina Sintase/metabolismo , Fungos/enzimologia , Hexosaminas/biossíntese , Modelos Biológicos , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse C alpha subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene.
Assuntos
Blastocladiella/enzimologia , Blastocladiella/genética , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/genética , Genes Fúngicos , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Primers do DNA , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , DNA Complementar/metabolismo , Éxons , Biblioteca Genômica , Íntrons , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
GTP gamma S stimulates adenylyl cyclase in particulate fractions of Blastocladiella emersonii zoospores. Cholera toxin catalyses the ADP-ribosylation of a membrane protein of a molecular weight (46,000) similar to that of the alpha subunit of Gs found in vertebrate cells. A membrane protein of 46 kDa can also be recognized in Western blots by an antipeptide antiserum (RM/1) raised against the C-terminus of G alpha 2-subunits. These results suggest that a G-protein mediates the regulation of Blastocladiella adenylyl cyclase by guanine nucleotides.
Assuntos
Adenilil Ciclases/metabolismo , Blastocladiella/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/metabolismo , Difosfato de Adenosina/metabolismo , Inibidores de Adenilil Ciclases , Blastocladiella/fisiologia , Western Blotting , Temperatura Alta , Immunoblotting , Esporos FúngicosRESUMO
Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.
Assuntos
Blastocladiella/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , Fosfoproteínas Fosfatases/metabolismo , Blastocladiella/enzimologia , Blastocladiella/crescimento & desenvolvimento , Parede Celular/metabolismo , Quitina/metabolismo , Éteres Cíclicos/farmacologia , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Esporos Fúngicos/enzimologia , Esporos Fúngicos/metabolismoRESUMO
We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene.