Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The a subunit is a membrane component of the F1F0-ATP synthase from Escherichia coli. Regions of a which appear important for membrane insertion or F0 assembly have been identified by analysis of both deletion mutants and fusion proteins which link the mutant a subunits to alkaline phosphatase. This analysis suggests the hydrophilic, amino-terminal domain of a is required for proper membrane targeting and/or insertion of the nascent polypeptide. In addition, the subcellular fractionation of four different a subunit-beta-galactosidase fusion proteins suggests this domain is localized to the periplasm, in agreement with a proposed topological model of the protein (Lewis, M.J., Chang, J.A., and Simoni, R.D. (1990) J. Biol. Chem. 265, 10541-10550). Deletions within the next three putative loops of a appear to have no significant effect on membrane targeting or insertion. Rather, they seem to interfere with the subsequent assembly of a functional enzyme.
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PMID:Deletions in hydrophilic domains of subunit a from the Escherichia coli F1F0-ATP synthase interfere with membrane insertion or F0 assembly. 153 41

Phosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a lambda gt11 expression library with antibody raised against the mitochondrial Ca(2+)-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498-11503, 1988). Similar clones were obtained by the immunoscreening of a rat heart expression library. In agreement with others, the open reading frames of the cDNAs from the two species coded for the same amino acid sequence. No difference in S6 of the human neoplastic cells compared to that of non-neoplastic cells was found. However, common antigenic determinants in S6 and CaBI were indicated. Accordingly, S6 was purified from rat liver ribosomes and antiserum prepared. Immuno-dot blot and Western blot analyses showed high specific reactivity between S6, the cloned chimeric beta-galactosidase fusion protein from a cDNA clone, and CaBI with anti-S6 and anti-CaBI antibodies. The antibodies also showed a high degree of discrimination for S6 and CaBI. Neither interacted with the other ribosomal proteins nor with another ATPase inhibitor protein from bovine heart mitochondria. Neither interacted with the Ca(2+)-binding proteins, calmodulin, oncomodulin, Protein C, or Factor X. Prothrombin was weakly reactive with anti-CaBI but not with anti-S6. Thus, the results fulfill the specific criteria for the concept and operational definition of common protein epitopes in S6 and CaBI. However, neither prothrombin nor S6 fusion protein inhibited mitochondrial ATPase activity even at 20 times the concentrations at which CaBI gave 97% inhibition.
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PMID:Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria. 183 89

The respiratory deficiency of two noncomplementing mutants of Saccharomyces cerevisiae (C41 and N28) has been shown to be due to mutations in HEM2, the structural gene for delta-aminolevulinate dehydratase. The mutants are unable to convert delta-aminolevulinic acid to porphobilinogen and are not complemented by the hem2 mutant GL4 (Gollub, E. G., Liu, K.-P., Dagan, J., Adlersberg, M., and Sprinson, D. B. (1977) J. Biol. Chem. 252, 2846-2854). A gene capable of complementing the respiratory deficiency of C41 and N28 has been cloned by transformation of a hem2 mutant with a recombinant plasmid library of wild type yeast nuclear DNA. The sequence of the protein encoded by the cloned gene exhibits extensive homology to the recently reported sequence of human delta-aminolevulinate dehydratase (Wetmur, J. G., Bishop, D. F., Cantelmo, C., and Desnick, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7703-7707). Several approaches were taken to study the effect of heme on transcription of PET genes known to code for subunit components of respiratory enzymes and of mitochondrial ATPase. The first involved measurements of the steady state levels of mRNAs for subunit 5 of cytochrome oxidase and the beta subunit of F1 ATPase in wild type and in a hem2 mutant. Secondly, transcription of the genes coding for the cytochrome oxidase and ATPase subunits as well as of the COR1 gene coding for the 44-kDa core 1 subunit of coenzyme QH2-cytochrome c reductase was quantitated by fusing the 5'-flanking and part of the coding region of each gene to the lacZ gene of Escherichia coli in vectors capable of integrating into yeast chromosomal DNA. The different lacZ fusions were integrated into nuclear DNA of a wild type strain and of hem2 mutants allowing expression of beta-galactosidase to be studied as a function of intracellular heme. These experiments indicate that the promoters of the genes for subunits of the respiratory complexes are regulated by heme. In contrast, the expression of the ATPase subunit appears to be heme-independent. Because neither subunit 5 of cytochrome oxidase nor the core 1 subunit of coenzyme QH2-cytochrome c reductase are hemoproteins, transcriptional regulation by heme may be a general mechanism for controlling the synthesis of mitochondrial proteins involved in respiration.
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PMID:Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and COR1 by heme. 244 51

The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The beta-subunit-beta-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.
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PMID:Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. 633 Jul 27

Subunit interactions among the F1-ATPase subunits were studied by the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Escherichia coli H+-ATPase fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of the alpha and beta subunit genes, and of the epsilon and gamma subunit genes showed high levels of reporter gene expression, while those of alpha and delta, beta and delta, gamma and delta, and delta and epsilon demonstrated weak but significant reporter gene expression. However, combinations of alpha and gamma, beta and gamma, alpha and epsilon, and beta and epsilon did not induce reporter expression. None of the fused genes alone induced reporter gene expression. These results suggested that specific and strong interactions between the alpha and beta, gamma and epsilon, and weak interactions between the alpha and delta, beta and delta, and gamma and delta subunits occurred in yeast cells in the two-hybrid system. Effects of previously identified mutant beta subunits with Leu-40 to Pro. Glu-41 to Lys or Pro-332 to Gln substitutions which caused defects in molecular assembly of F1-ATPase were analyzed with regard to alpha-beta interactions. No interaction of the alpha and beta subunits was observed in this system using the beta subunit with mutation of Pro-332 to Gln. However, for the other two mutations, alpha-beta interactions were observed. This system may be useful for isolating mutants which have defects in interaction of F1-ATPase subunits.
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PMID:Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system. 864 96

The genes for the beta and epsilon subunits of maize chloroplast ATP synthase are encoded by the organelle genome, are cotranscribed, and have overlapping translation initiation and termination codons. To determine whether the atpB and atpE genes are translationally coupled, they were transformed into Escherichia coli on a multicopy plasmid. Synthesis of full-length beta and epsilon polypeptides demonstrated correct initiation of translation by the bacterial ribosomes. To assay for translational coupling, the promoter-distal atpE gene was fused to lacZ, resulting in the synthesis of an active hybrid beta-galactosidase. A frameshift mutation was introduced into the promoter-proximal atpB gene, and its effect on the transcription and translation of the atpE::lacZ fusion was measured. The mutation resulted in a 1000- to 2000-fold reduction in beta-galactosidase activity, but only a 2-fold decrease in LacZ mRNA synthesis rates or galactoside transacetylase levels. Similar results were obtained when the atpB/atpE::lacZ fusion and the atpB frameshift mutation were introduced into the photosynthetic cyanobacterium Synechocystis sp. PCC6803. We show that >99% of atpE translation depends on successful translation of atpB and, thus, conclude that the two genes are translationally coupled.
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PMID:Translational coupling of the maize chloroplast atpB and atpE genes. 1659 48

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding beta-galactosidase was detected. Of all the combinations, that of gamma and epsilon subunit genes showed the highest level of reporter gene expression, while those of alpha and beta, a and epsilon, beta and epsilon and beta and delta induced stable and significant reporter gene expression. The combination of delta and epsilon as well as that of delta and gamma induced weak and unstable reporter gene expression. However, combinations of alpha and gamma, beta and gamma and alpha and delta did not induce reporter gene expression. These results suggested that specific and strong interactions between gamma and epsilon, alpha and beta, alpha and epsilon, beta and epsilon and beta and delta subunits, and weak and transient interactions between delta and epsilon and delta and gamma subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.
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PMID:Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea. 1872 69

PAS kinase 1 (Psk1) is a key regulator of respiration in Saccharomyces cerevisiae Herein the molecular mechanisms of this regulation are explored through the characterization of its substrate, Centromere binding factor 1 (Cbf1). CBF1-deficient yeast displayed a significant decrease in cellular respiration, while PAS kinase-deficient yeast, or yeast harboring a Cbf1 phosphosite mutant (T211A) displayed a significant increase. Transmission electron micrographs showed an increased number of mitochondria in PAS kinase-deficient yeast consistent with the increase in respiration. Although the CBF1-deficient yeast did not appear to have an altered number of mitochondria, a mitochondrial proteomics study revealed significant differences in the mitochondrial composition of CBF1-deficient yeast including altered Atp3 levels, a subunit of the mitochondrial F₁-ATP synthase complex. Both beta-galactosidase reporter assays and western blot analysis confirmed direct transcriptional control of ATP3 by Cbf1 In addition, we confirmed the regulation of yeast lipid genes LAC1 and LAG1 by Cbf1 The human homolog of Cbf1, Upstream transcription factor 1 (USF1), is also known to be involved in lipid biogenesis. Herein, we provide the first evidence for a role of USF1 in respiration since it appeared to complement Cbf1 in vivo as determined by respiration phenotypes. In addition, we confirmed USF1 as a substrate of human PAS kinase (hPASK) in vitro Combined, our data supports a model in which Cbf1/USF1 functions to partition glucose toward respiration and away from lipid biogenesis, while PAS kinase inhibits respiration in part through the inhibition of Cbf1/USF1.
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PMID:The Regulation of Cbf1 by PAS Kinase Is a Pivotal Control Point for Lipogenesis vs. Respiration in Saccharomyces cerevisiae. 3038 Dec 92

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