RESUMO
The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH and complex II) of the respiratory chain is highly conserved in evolution [Gould et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1934-1938]. We have cloned the entire human Ip cDNA, as well as the Ip-encoding gene (SDH-B) from two genomic human libraries. The cDNA contains a coding sequence of 840 nt, flanked by a 5'-UTR of 133 nt and a 3'-UTR of 123 nt. The entire transcript is encoded by eight exons within approx. 40 kb. The seven introns range in size from 0.75 kb to > 11 kb, and they appear to be of the 'late' intron class. Approx. 5 kb of upstream sequence was also cloned, and approx. 2.4 kb of the promoter region were sequenced and analyzed for consensus elements binding potential transcription factors and transcriptional activators.
Assuntos
Proteínas Ferro-Enxofre/genética , Mitocôndrias/enzimologia , Succinato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Éxons/genética , Biblioteca Genômica , Humanos , Íntrons/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Conformação Proteica , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosAssuntos
Carboxina/toxicidade , Resistência Microbiana a Medicamentos/genética , Genes Fúngicos , Proteínas Ferro-Enxofre/biossíntese , Succinato Desidrogenase/biossíntese , Ustilago/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Proteínas Ferro-Enxofre/genética , Dados de Sequência Molecular , Especificidade da Espécie , Succinato Desidrogenase/antagonistas & inibidores , Succinato Desidrogenase/genética , Ustilago/efeitos dos fármacos , Ustilago/genética , Zea mays/microbiologiaRESUMO
A series of chimeric human/yeast IP genes were constructed in order to investigate domains of the iron-sulfur protein (IP) that are important for assembly and/or activity of complex II of the electron transport system in Saccharomyces cerevisiae. These genes were expressed in a respiration-deficient yeast mutant in which the endogenous IP gene had been disrupted. Substitutions at the N-terminus were tolerable. Substituting the region covering the first iron-sulfur center [2Fe-2S] had no effect on assembly, while activity decreased 2-5-fold. The addition of seven amino acids from the human peptide, including four charged residues, at the C-terminus did not perturb either assembly or activity. A region between the first and second cysteine clusters was identified which when substituted caused a complete failure in the assembly of complex II. It includes a 15 amino acid stretch which shows the greatest variability between species. Larger substitutions including this segment failed as well. Exchanging the region between the second and third cysteine clusters making up the [4Fe-4S] and [3Fe-4S] centers enabled transformants to grow on nonfermentable carbon sources, yet no SDH activity was observed in vitro. The IP and FP proteins accumulate to wild-type levels in these mutants. We speculate that the lack of observed activity is due to the lability of iron-sulfur centers in isolated, broken mitochondria.
Assuntos
Proteínas Ferro-Enxofre/biossíntese , Fígado/enzimologia , Mitocôndrias/enzimologia , Complexos Multienzimáticos/biossíntese , Oxirredutases/biossíntese , Saccharomyces cerevisiae/enzimologia , Succinato Desidrogenase/biossíntese , Sequência de Aminoácidos , Animais , Northern Blotting , Bovinos , Clonagem Molecular , Transporte de Elétrons , Complexo II de Transporte de Elétrons , Humanos , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredutases/metabolismo , Consumo de Oxigênio , Mutação Puntual , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Succinato Desidrogenase/metabolismoRESUMO
The sequence of an allele encoding the iron-sulphur protein (Ip) subunit of succinate dehydrogenase (Sdh) was determined following PCR amplification of genomic DNA from a carboxin (Cbx)-sensitive Ustilago maydis strain. Comparison of this sequence with that of the Ip allele from a Cbx-resistant strain (IPr) revealed a two-base difference between the sequences. This mutation led to the substitution of a leucine residue for a histidine residue within the third cysteine-rich cluster of the deduced amino-acid sequence of the Ipr allele. This cluster, which is associated with the S3 iron-redox centre, is involved in the transport of electrons from succinate to ubiquinone (Q). Confirmation that this nucleotide change led to enhanced resistance to Cbx was obtained following mutagenesis of the sensitive Ip allele to the resistant form and expression of the mutated allele in U. maydis.
