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Michael Thomm - One of the best experts on this subject based on the ideXlab platform.
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the rpb7 orthologue e is required for transcriptional activity of a reconstituted archaeal Core Enzyme at low temperatures and stimulates open complex formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:Abstract RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E′F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 °C but was almost inactive at 60 °C. E′ stimulated the activity of the Core Enzyme at 60 °C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E′, and of archaeal TFE to open complex formation. Opening in the –2 and –4 region could be achieved by the Core Enzyme, subunit E′ stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E′-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
The Journal of biological chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
Souad Naji - One of the best experts on this subject based on the ideXlab platform.
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the rpb7 orthologue e is required for transcriptional activity of a reconstituted archaeal Core Enzyme at low temperatures and stimulates open complex formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:Abstract RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E′F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 °C but was almost inactive at 60 °C. E′ stimulated the activity of the Core Enzyme at 60 °C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E′, and of archaeal TFE to open complex formation. Opening in the –2 and –4 region could be achieved by the Core Enzyme, subunit E′ stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E′-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
The Journal of biological chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
Sebastian Grünberg - One of the best experts on this subject based on the ideXlab platform.
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the rpb7 orthologue e is required for transcriptional activity of a reconstituted archaeal Core Enzyme at low temperatures and stimulates open complex formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
Journal of Biological Chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:Abstract RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E′F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 °C but was almost inactive at 60 °C. E′ stimulated the activity of the Core Enzyme at 60 °C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E′, and of archaeal TFE to open complex formation. Opening in the –2 and –4 region could be achieved by the Core Enzyme, subunit E′ stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E′-independent role of TFE in the stabilization of open complexes.
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The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation
The Journal of biological chemistry, 2007Co-Authors: Souad Naji, Sebastian Grünberg, Michael ThommAbstract:RNA polymerases from Archaea and Eukaryotes consist of a Core Enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the Core Enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The Core Enzyme showed significant activity between 70 and 80 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the Core Enzyme at 60 degrees C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the Core Enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the -2 and -4 region could be achieved by the Core Enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.
Akira Ishihama - One of the best experts on this subject based on the ideXlab platform.
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The interaction between sigmaS, the stationary phase sigma factor, and the Core Enzyme of Escherichia coli RNA polymerase.
Genes to cells : devoted to molecular & cellular mechanisms, 2002Co-Authors: Frédéric Colland, Akira Ishihama, Nobuyuki Fujita, Annie KolbAbstract:Background: The RNA polymerase holoEnzyme of Escherichia coli is composed of a Core Enzyme (subunit structure α2ββ′) associated with one of the σ subunits, required for promoter recognition. Different σ factors compete for Core binding. Among the seven σ factors present in E. coli, σ70 controls gene transcription during the exponential phase, whereas σS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled σS and σ70, we compared the affinities of both σ factors for Core binding and investigated the structural changes in the different subunits involved in the formation of the holoEnzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that σS binds to the Core Enzyme with fivefold reduced affinity compared to σ70. Using iron chelate protein footprinting, we show that the Core Enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of σS, while increasing the accessibility in region 4.1 of σS. We have also analysed the positioning of σS on the holoEnzyme by the proximity-dependent protein cleavage method using σS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the β and β′ subunits, but not α. Regions 2.5, 3.1 and 3.2 of σS are close to both β and β′ subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using σS and previous data from σ70 indicates similar contact patterns on the Core subunits and similar characteristic changes associated with holoEnzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.
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Competition among seven Escherichia coli σ subunits: relative binding affinities to the Core RNA polymerase
Nucleic acids research, 2000Co-Authors: Hiroto Maeda, Nobuyuki Fujita, Akira IshihamaAbstract:Seven different species of the RNA polymerase sigma subunit exist in Escherichia coli, each binding to a single species of the Core Enzyme and thereby directing transcription of a specific set of genes. To test the sigma competition model in the global regulation of gene transcription, all seven E.coli sigma subunits have been purified and compared for their binding affinities to the same Core RNA polymerase (E). In the presence of a fixed amount of sigma(70), the principal sigma for growth-related genes, the level of Esigma(70) holoEnzyme formation increased linearly with the increase in Core Enzyme level, giving an apparent K:(d) for the Core Enzyme of 0.26 nM. Mixed reconstitution experiments in the presence of a fixed amount of Core Enzyme and increasing amounts of an equimolar mixture of all seven sigma subunits indicated that sigma(70) is strongest in terms of Core Enzyme binding, followed by sigma(N), sigma(F), sigma(E)/sigma(FecI), sigma(H) and sigma(S) in decreasing order. The orders of Core binding affinity between sigma(70) and sigma(N) and between sigma(70) and sigma(H) were confirmed by measuring the replacement of one Core-associated sigma by another sigma subunit. Taken together with the intracellular sigma levels, we tried to estimate the number of each holoEnzyme form in growing E. coli cells.
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Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Jeffrey T. Owens, Akira Ishihama, Nobuyuki Fujita, Reiko Miyake, Katsuhiko S. Murakami, Albert J. Chmura, Claude F. MearesAbstract:The Core Enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the Core Enzyme subunits (α2ββ′), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ70 was bound to the Core Enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71–75], the cut sites from different probe-modified σ70 proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).
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Promoter Selectivity Control of RNA Polymerase
Mechanisms of Transcription, 1997Co-Authors: Akira IshihamaAbstract:The RNA polymerase (RPase) holoEnzyme of Escherichia coli is composed of a Core Enzyme with the subunit structure α2ββ′, combined with one of the multiple species of σ subunits, which provides the recognition activity for two hexanucleotide sequences of promoters, generally located near -35 and -10 positions relative to the transcription start site. The Core Enzyme carries all the functions necessary for RNA polymerization but the σ subunit is required for transcription initiation from promoters. Each σ subunit recognizes a different set of promoters and therefore the promoter selectivity of RPase can be modulated by replacement of the σ subunit (Helmann and Chamberlin 1988; Ishihama 1988). At present, seven different molecular species of σ subunit are known to exist in E. coli.
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[9] Reconstitution of RNA polymerase
Methods in enzymology, 1996Co-Authors: Nobuyuki Fujita, Akira IshihamaAbstract:Publisher Summary The DNA-dependent RNA polymerase of Escherichia coli is composed of Core Enzyme and one of at least six molecular species of the σ subunit. Despite the complexity of its subunit composition and its extraordinarily large molecular size, RNA polymerase is one of the few large molecular assemblies that can be successfully reconstituted from isolated individual subunits. Core Enzyme can be reconstituted by mixing α, β, and β' subunits under denaturing conditions, and then removing the denaturant by dialysis against the appropriate reconsitution buffer. The Core Enzyme reconstituted at low temperature is known to exist as an inactive premature from. It can be activated by heating at 30 ° for 30–60 min. Although the activated Core Enzyme can be used directly for many purposes, it contains considerable amounts of free subunits and α 2 β subassembly. For quantitative experiments, therefore, further purification of the reconstituted Enzyme is essential.
Gernot Walter - One of the best experts on this subject based on the ideXlab platform.
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The formation and activity of PP2A holoEnzymes do not depend on the isoform of the catalytic subunit
The Journal of biological chemistry, 2002Co-Authors: Jin Zhou, Huong T Pham, Gernot WalterAbstract:Abstract The protein phosphatase 2A holoEnzyme is composed of one catalytic C subunit, one regulatory/scaffolding A subunit, and one regulatory B subunit. The Core Enzyme consists of A and C subunits only. The A and C subunits both exist as two closely related isoforms, α and β. The B subunits belong to four weakly related or unrelated families, designated B, B′, B", and B‴, with multiple members in each family. The existence of two A and two C subunit isoforms permits the formation of four Core Enzymes, AαCα, AαCβ, AβCα, and AβCβ, and each Core Enzyme could in theory give rise to multiple holoEnzymes. Differences between Cα and Cβ in expression and subcellular localization during early embryonic development have been reported, which imply that Cα and Cβ have different functions. To address the question of whether these differences might be caused by enzymatic differences between Cα and Cβ, we purified six holoEnzymes composed of AαCα or AαCβ Core Enzyme and B subunits from the B, B′, or B" families. In addition, we purified four holoEnzymes composed of AβCα or AβCβ and B′α1 or B"/PR72. The phosphatase activity of each purified form was assayed using myelin basic protein and histone H1 as substrates. We found that Cα and Cβ have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Cα and Cβ in binding A or B subunits. These data suggest that the distinct functions of Cα and Cβ are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Cα and Cβ are discussed.
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Dilated cardiomyopathy in transgenic mice expressing a mutant A subunit of protein phosphatase 2A.
American journal of physiology. Heart and circulatory physiology, 2000Co-Authors: Neil Brewis, Kim Ohst, Katherine Fields, Antonio Rapacciuolo, Danny M. Chou, Colin M. Bloor, Wolfgang H. Dillmann, Howard A. Rockman, Gernot WalterAbstract:The protein phosphatase 2A (PP2A) holoEnzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B. The PP2A Core Enzyme is composed of subunits A and C. Both the holoEnzyme and the Core Enzyme are similarly abundant in heart tissue. Transgenic mice were generated expressing high levels of a dominant negative mutant of the A subunit (A delta 5) in the heart, skeletal muscle, and smooth muscle that competes with the endogenous A subunit for binding the C subunit but does not bind B subunits. We found that the ratio of Core Enzyme to holoEnzyme was increased in A delta 5-expressing hearts. Importantly, already at day 1 after birth, A delta 5-transgenic mice had an increased heart weight-to-body weight ratio that persisted throughout life. Echocardiographic analysis of A delta 5-transgenic hearts revealed increased end-diastolic and end-systolic dimensions and decreased fractional shortening. In addition, the thickness of the septum and of the left ventricular posterior wall was significantly reduced. On the basis of these findings, we consider the heart phenotype of A delta 5-transgenic mice to be a form of dilated cardiomyopathy that frequently leads to premature death.
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Binding Specificity of Protein Phosphatase 2A Core Enzyme for Regulatory B Subunits and T Antigens
Journal of virology, 1999Co-Authors: Ralf Ruediger, Katherine Fields, Gernot WalterAbstract:The Core Enzyme of protein phosphatase 2A is composed of a regulatory subunit A and a catalytic subunit C. It is controlled by three types of regulatory B subunits (B, B′, and B") and by tumor (T) antigens, which are unrelated by sequence but bind to overlapping regions on the A subunit. To find out whether the different B subunits and T antigens bind to identical or distinct amino acids of the A subunit, mutants were generated and their abilities to bind B subunits and T antigens were tested. We found that some amino acids are involved in the binding of all types of B subunits, whereas others are specifically involved in the binding of one or two types of B subunits. T-antigen-binding specificity does not correlate with that of a particular type of B subunit.
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Increasing the Ratio of PP2A Core Enzyme to HoloEnzyme Inhibits Tat-Stimulated HIV-1 Transcription and Virus Production☆
Virology, 1997Co-Authors: Ralf Ruediger, Kim Ohst, Neil Brewis, Gernot WalterAbstract:Abstract We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoEnzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) Core Enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A Core and holoEnzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, AΔ5, which binds the C but not the B subunit. This resulted in an increase in the amount of Core Enzyme and a decrease in the amount of holoEnzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant AΔ5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant AΔ5. These results demonstrate that the balance between PP2A holoEnzyme and Core Enzyme is important for HIV-1 gene expression and virus production.
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separation of pp2a Core Enzyme and holoEnzyme with monoclonal antibodies against the regulatory a subunit abundant expression of both forms in cells
Molecular and Cellular Biology, 1997Co-Authors: E Kremmer, Kim Ohst, J Kiefer, Neil Brewis, Gernot WalterAbstract:Protein phosphatase 2A (PP2A) holoEnzyme is composed of a catalytic subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, Core Enzyme, is composed only of subunits A and C. It is generally believed that Core Enzyme does not exist in cells but is an artifact of Enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and Core Enzyme but not holoEnzyme. Another antibody, 6G3, recognized an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the A alpha subunit (designated 6F9) precipitated free A subunit, Core Enzyme, and holoEnzyme. 6F9, but not 5H4, recognized holoEnzymes containing either B, B', or B" subunits. These results demonstrate that B subunits from three unrelated gene families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoEnzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, Core Enzyme and holoEnzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoEnzyme, Core Enzyme represented at least one-third of the total PP2A. We conclude that Core Enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.