RESUMO
Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.
Assuntos
Cálcio/farmacologia , Frutose-Bifosfatase/antagonistas & inibidores , Ácido Glutâmico/genética , Músculo Esquelético/enzimologia , Monofosfato de Adenosina/farmacologia , Substituição de Aminoácidos/genética , Animais , Cátions Bivalentes/farmacologia , Frutose-Bifosfatase/química , Frutose-Bifosfatase/genética , Ácido Glutâmico/química , Glutamina/química , Glutamina/genética , Mutação Puntual , CoelhosRESUMO
All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1-like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H(2)O(2) treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1-like proteins respond to metalloids through distinct mechanisms.
Assuntos
Antimônio/farmacologia , Arsênio/farmacologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Arseniato Redutases , ATPases Transportadoras de Arsenito , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Núcleo Celular/ultraestrutura , Cisteína/genética , Cisteína/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Bombas de Íon/genética , Bombas de Íon/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Elementos de Resposta/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.