ABSTRACT
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.
Subject(s)
Caprylates/pharmacology , Gene Expression Regulation, Enzymologic , Genes, Fungal , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/enzymology , Cell Membrane/enzymology , Cloning, Molecular , Gene Expression Regulation, Enzymologic/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & developmentABSTRACT
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.
Subject(s)
Adenosine Triphosphate/metabolism , Peptides/isolation & purification , Proton-Translocating ATPases/chemistry , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphate/analogs & derivatives , Amino Acid Sequence , Base Sequence , Cell Membrane/enzymology , Cloning, Molecular , Escherichia coli , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Glutathione Transferase/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptides/analysis , Peptides/metabolismSubject(s)
Fungal Proteins/genetics , Open Reading Frames , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , DNA, Fungal/chemistry , Diploidy , Fungal Proteins/chemistry , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Repetitive Sequences, Nucleic Acid , Zinc Fingers/geneticsABSTRACT
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.
Subject(s)
Chromosomes, Fungal/chemistry , DNA, Fungal/chemistry , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Chromosome Mapping , Cloning, Molecular , Cosmids , DNA, Fungal/genetics , Mutation , Open Reading Frames , Restriction Mapping , Saccharomyces cerevisiae/ultrastructureSubject(s)
Fungal Proteins/genetics , Leucine Zippers , Membrane Proteins/genetics , Open Reading Frames , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , DNA, Fungal/chemistry , DNA, Fungal/genetics , Fungal Proteins/chemistry , Membrane Proteins/chemistry , Molecular Sequence Data , Mutagenesis , Repetitive Sequences, Nucleic AcidSubject(s)
Carrier Proteins/genetics , Fungal Proteins/genetics , Open Reading Frames , Oxidoreductases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Carrier Proteins/chemistry , DNA, Fungal/chemistry , Fungal Proteins/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Saccharomyces cerevisiae/physiology , Spores, Fungal , TATA BoxABSTRACT
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.
Subject(s)
Genes, Fungal , Saccharomyces cerevisiae/genetics , Suppression, Genetic , Amino Acid Sequence , Base Sequence , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , Genes, Dominant , Genes, Lethal , Molecular Sequence Data , Mutation , Plasmids , RNA, Fungal/genetics , RNA, Fungal/isolation & purification , Restriction Mapping , Saccharomyces cerevisiae/growth & development , Transformation, GeneticABSTRACT
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.
Subject(s)
DNA-Binding Proteins/physiology , Fungal Proteins/physiology , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/physiology , DNA Mutational Analysis , Gene Expression Regulation , Glycolysis , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Fungal/genetics , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Transcription, GeneticABSTRACT
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.