RESUMEN
A peak of plasma membrane H(+)-ATPase activity during exponential growth is correlated with the expression of the PMA1 gene as monitored by measurements of the beta-galactosidase activity from a PMA1-lacZ fusion. This peak of activity is also correlated to the content of the H(+)-ATPase protein in yeast plasma membrane as shown by quantitative immunodetection. The PMA2-lacZ fusion assay indicates that the expression of the PMA2 gene is activated somewhat later during exponential phase but under all circumstances its activity remains at least 500-fold lower than that of the PMA1-lacZ fusion. A slight but significant stimulation of ATPase activity by low concentrations of octanoic acid coincides with a decrease in the PMA1 gene expression. It is concluded that octanoic acid stimulates de PMA1 ATPase activity by posttranslational mechanisms.
Asunto(s)
Caprilatos/farmacología , Regulación Enzimológica de la Expresión Génica , Genes Fúngicos , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/enzimología , Membrana Celular/enzimología , Clonación Molecular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli. The GST-L peptide contained the hydrophilic region from Ala340 to Ser660. The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E. coli culture. The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL. ATP competitively displaced the TNP-ATP binding. The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein. The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide. Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5.
Asunto(s)
Adenosina Trifosfato/metabolismo , Péptidos/aislamiento & purificación , ATPasas de Translocación de Protón/química , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfato/análogos & derivados , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/enzimología , Clonación Molecular , Escherichia coli , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Péptidos/análisis , Péptidos/metabolismoRESUMEN
A physical map of a contiguous DNA fragment of 60 kb, extending from the centromere to TRP5 on the left arm of the chromosome VII of Saccharomyces cerevisiae, strain IL125-2B, was established. Within a 31 kb region from PMA1 towards TRP5, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified in cells grown exponentially on rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2.3 kb average length, was detected every 2.7 kb of DNA. The physical and genetical distances between the markers CEN7, pma1, leu1, pdr1 and trp5 were compared. A recombination frequency of 1 cM corresponds to an average distance of 3.3 kb between alleles in this region of chromosome VII.
Asunto(s)
Cromosomas Fúngicos/química , ADN de Hongos/química , Saccharomyces cerevisiae/genética , Transcripción Genética , Mapeo Cromosómico , Clonación Molecular , Cósmidos , ADN de Hongos/genética , Mutación , Sistemas de Lectura Abierta , Mapeo Restrictivo , Saccharomyces cerevisiae/ultraestructuraAsunto(s)
ADN de Hongos/química , Farmacorresistencia Microbiana/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Desoxirribonucleasa HindIII , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mapeo Restrictivo , Programas InformáticosAsunto(s)
Proteínas Fúngicas/genética , Leucina Zippers , Proteínas de la Membrana/genética , Sistemas de Lectura Abierta , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis , Secuencias Repetitivas de Ácidos NucleicosAsunto(s)
Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Sistemas de Lectura Abierta , Oxidorreductasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , ADN de Hongos/química , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas , TATA BoxAsunto(s)
Proteínas Fúngicas/genética , Sistemas de Lectura Abierta , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/química , Diploidia , Proteínas Fúngicas/química , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Secuencias Repetitivas de Ácidos Nucleicos , Dedos de Zinc/genéticaRESUMEN
In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.
Asunto(s)
Genes Fúngicos , Saccharomyces cerevisiae/genética , Supresión Genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Genes Dominantes , Genes Letales , Datos de Secuencia Molecular , Mutación , Plásmidos , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Transformación GenéticaRESUMEN
The H+-ATPase, located in the yeast plasma membrane and encoded by the PMA1 gene, provides energy for the active transport of nutrients and regulates intracellular pH. Expression of the PMA1 gene is essential for cell growth and development. In this study, progressive deletions of the PMA1 promoter fused to the beta-galactosidase gene have identified two upstream activating sequences. These upstream activating sequences have high homologies with the consensus sequence known to control the expression of the ribosomal protein genes (RPG). In vivo deletion of these RPG sequences from the PMA1 gene results in slower growth and reduces ATPase activity to one-third of its original value. The RPG sequences from PMA1 interact with the promoter binding factor TUF. Thus, PMA1 belongs to the RPG-TUF system which includes many constitutive genes encoding nonrelated functions such as ATP metabolism, transcription, translation, and active transport.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , Análisis Mutacional de ADN , Regulación de la Expresión Génica , Glucólisis , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN de Hongos/genética , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The Saccharomyces cerevisiae gene PDR1, responsible for pleiotropic drug resistance, was isolated from a genomic DNA cosmid library by hybridization to the flanking LEU1 gene, followed by subcloning the drug-sensitive phenotype into the transformed pdr1-1, pdr1-2, and pdr1-3 drug-resistant mutants. A RNA molecule of 3.5 kilobases was identified as the PDR1 transcript. The nucleotide sequence of the complementing DNA fragment contained a 3192-nucleotide open reading frame. Disruption of the pdr1 and PDR1 genes restored or increased drug sensitivity. Analysis of the PDR1 deduced amino acid sequence revealed several homologies to four different regulatory proteins involved in the control of gene expression, including a cysteine-rich motif suggested to be a metal-binding domain for DNA recognition. A model is proposed of a general transcriptional control by PDR1 of several target genes encoding proteins from plasma, mitochondria, and possibly other permeability barriers.