RESUMO
We have expressed the pore-forming subunits (Kir 6.1 and Kir 6.2) of the mammalian ATP-sensitive potassium channel in a potassium-transport deficient yeast strain (trk1 trk2). Functional expression of Kir 6.2 and Kir 6.1 can complement growth deficiency weakly and strongly respectively of the yeast strain on low-potassium medium. Mutations of Kir 6.2 that abolish ATP sensitivity (K185Q, I182Q) and enhance trafficking to the plasma membrane surface (Kir 6.2DeltaC36) lead to significantly better growth rescue. Growth rescue of Kir 6.1, Kir 6.2 and the above mutants can be inhibited by pharmacological agents (cesium ions, phentolamine and quinine) known to decrease channel activity by direct interaction with the pore forming subunit. Thus we have developed a system in yeast that can report both loss and gain of function mutations in these subunits and pharmacological interventions.
Assuntos
Expressão Gênica/genética , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/farmacologia , Substituição de Aminoácidos/genética , Animais , Césio/farmacologia , Cloretos/farmacologia , Teste de Complementação Genética , Vetores Genéticos/genética , Mutação/genética , Fentolamina/farmacologia , Potássio/metabolismo , Potássio/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Quinina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.
Assuntos
Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Genes Supressores , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Xenopus laevisRESUMO
More than 80% of the extracellular Avicelase, endoglucanase, xylanase and mannanase activities of the anaerobic fungus Piromyces were associated with a cellulose-binding complex. The complex was composed of at least 10 polypeptides ranging in size from 190 kDa to 50 kDa, and contained numerous endoglucanases, xylanases and mannanases. Multiple genes encoding each of these activities were isolated from an expressing cDNA library.
