RESUMO
Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).
Assuntos
Citrato (si)-Sintase/metabolismo , Peroxissomos/enzimologia , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Citrato (si)-Sintase/química , Citrato (si)-Sintase/genética , Primers do DNA , DNA Fúngico , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The coding region of the mitochondrial citrate synthase gene (CIT1) from Saccharomyces cerevisiae was amplified by PCR and cloned into an expression vector (pAL4) downstream of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans to yield pALCS1. Transformation of A. nidulans A773 with this construct gave stable transformants, AYC#1 and AYC#2, that were phenotypically stable for several mitotic divisions. Southern blot analysis showed that the CIT1 gene was successfully integrated into the chromosomes of the transformants. Western blot analysis and enzymatic assay for citrate synthase revealed that the integrated yeast gene was subject to inducible expression controlled by alcA promoter, which can be induced by threonine.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Citrato (si)-Sintase/biossíntese , Proteínas Fúngicas/biossíntese , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Southern Blotting , Citrato (si)-Sintase/genética , Indução Enzimática/genética , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos , Vetores Genéticos/síntese química , Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
We isolated a citrate synthase gene (citA) from Aspergillus nidulans. By analysis of the protein coding region, citA was shown to encode a citrate synthase (CitA) of 52.2 kDa consisting of 474 amino acid residues that were interrupted by seven introns. Also, the precursor CitA protein was revealed to have an N-terminal mitochondrial targeting signal of 35 amino acid residues containing an R-3 cleavage motif, R(32)-C-Y decreases S(35), which supports the fact that citA encodes the mitochondrial form of citrate synthase of A. nidulans. Southern blot analysis showed that citA is present as a single copy in the genome.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Citrato (si)-Sintase/genética , Genes Fúngicos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Fúngico/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Two different xylanases, CX-I and CX-II, from an alkalophilic fungus, Cephalosporium sp. strain RYM-202, have been purified to homogeneity. The enzymes had similar pH (7.5 to 8.0) and temperature (50(deg)C) optima and were stable over a wide pH range of 5.5 to 12.0. Both enzymes were shown to be cellulase-free endoxylanases with transglycosidation activity.
RESUMO
The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.