α, βI, βII, δ, and ε Protein Kinase C Isoforms and Compound Activity in the Sciatic Nerve of Normal and Diabetic Rats

Abstract: Defective protein kinase C (PKC) has been implicated in impaired Na⁺,K⁺-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, α, βI, βII, γ, δ, and ε isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. α, βI, βII, δ, and ε but no γ isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3–4 days after transection except for diminished βI and ε species; in Schwann cell cultures, only α, βII, δ, and ε were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na⁺,K⁺-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of δ, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6–12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Phorbol Ester-Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C-α in Human SH-SY5Y Neuroblastoma Cells

Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC₅₀ 0.6 µ M ) but not 100 m M K⁺-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K⁺-evoked release after pretreatment with 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) for 8 min, with IC₅₀ values of 0.7 and 2.4 µ M , respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µ M ), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K⁺-evoked NA release by >50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K⁺-evoked NA secretion is due to activation of PKC-\ga.     

Roles of protein kinase Cα isozyme in the regulation of oxidative stress and neuropeptide Y gene expression in phenylpropanolamine-mediated appetite suppression

Hypothalamic neuropeptide Y (NPY) is an appetite stimulant in the brain. Although regulation of NPY expression has been reported to contribute to the appetite-suppressing effect of phenylpropanolamine (PPA), it is still unknown if protein kinase C (PKC) is involved in this effect. Rats were daily treated with PPA for 4 days. Changes in food intake, hypothalamic NPY, PKC, and proopiomelanocortin (POMC) mRNA levels were assessed and compared. Results showed that the NPY gene was down-regulated following PPA treatment, which was parallel with the decrease of feeding. Moreover, several isotypes of PKC mRNA level (α, βI, βII, γ, δ, η, λ, ε, and ζ) were changed. Among these, α, δ, and λ PKC were up-regulated along with POMC gene expression which coincided with down-regulation of the NPY gene. To further determine if PKCα was involved, infusions of antisense oligonucleotide into the cerebroventricle were performed at 1 h before daily PPA treatment in free-moving rats. Results showed that PKCα knock-down could modify both anorexia induced by PPA and the NPY mRNA levels. Moreover, PKCα knock-down could also modify superoxide dismutase (SOD) gene expression. It is suggested that PKCα participates in the regulation of PPA-mediated appetite suppression via the modulation of NPY and SOD gene expression.     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Loss of Protein Kinase C-αβ in Brain of Heroin Addicts and Morphine-Dependent Rats

Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α₂-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.     

Activation of Protein Kinase C-α and Translocation of the Myristoylated Alanine-Rich C-Kinase Substrate Correlate with Phorbol Ester-Enhanced Noradrenaline Release from SH-SY5Y Human Neuroblastoma Cells

Abstract: The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the α-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-ε from cytosolic and membrane-associated to cytoskeleton- and membrane-associated TPA had no effect on the cytosolic location of PKC-ζ. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 µ M ). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 n M ) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-α (but not -ε or -ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-α.     

Nuclear Localization of the δ Subunit of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Cerebellar Granule Cells

Abstract : To examine the physiological roles of the δ subunit of Ca²⁺/calmodulin-dependent protein kinase ∥ (CaM kinase ∥δ) in brain, we examined the localization of CaM kinase ∥δ in the rat brain. A specific antibody to CaM kinase ∥δ1-δ4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the α, β, and γ subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase ∥δ1-δ4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products δ3.1, δ3.4, and δ3 that contained the nuclear localization signal. These CaM kinase ∥δ3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase ∥δ3 isoforms are involved in nuclear Ca²⁺ signaling in cerebellar granule cells.     

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-α and -β₁ were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-δ and -ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-β-phorbol or staurosporine, and that protein kinase C-ɛ is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-α and -ɛ decreased, and protein kinase C-β₁, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.     

Pb2+ reduces PKCs and NF-κB in vitro

The mechanism of lead (Pb²⁺)-induced neurotoxicity has not yet been fully elucidated. The purpose of this study was to examine the effects of Pb²⁺ on several protein kinase C (PKC) isoforms and the nuclear factor-κB (NF-κB)–I-κB kinase-alpha (IKK-α) axis in cultured neuronal cells. Neurons were isolated from rat fetal brain at the 18th day of gestation of pregnant Sprague Dawley rats and cultured for 10 days before use. Neurons were exposed to Pb²⁺ at concentrations of 10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ mol/L for 14 h and antigens of typical PKC-α,β,γ; novel PKC (ε, δ), atypical PKC (λ), NF-κB (p50), and IKK-α were enriched by immunoprecipitation and determined by western blotting. Total, calcium-dependent and independent PKC activities were also determined by counting the transferred γ-³² P in the substrate-histone. The results indicated that inorganic Pb²⁺ significantly reduced all PKC isoforms (α,β,γ, ε, λ) except δ, inhibiting the total, calcium-dependent and calcium-independent PKC activities in a dose-dependent manner. Additionally, Pb²⁺ gradually reduced NF-κB (p50) and IKK-α protein levels. This suggests that Pb²⁺ exhibits varying preference for individual PKC isoforms but reduces the NF-κB–IKK-α axis to a similar extent.     

Monogalactosyl Diglyceride, a Marker for Myelination, Activates Oligodendroglial Protein Kinase C

Abstract: Protein kinase C (PKC) is activated by 1,2- sn -diacylglycerol (DAG), the source of which can either be phosphatidylinositol bisphosphate or phosphatidylcholine. Here, we show that monogalactosyl diglyceride (MGDG), a minor galactolipid present in oligodendrocytes (OLs) and myelin, which is designated as a marker for myelination, can enhance OL PKC activity. Based on different calcium and substrate requirements we conclude that MGDG and DAG activate different isoforms of PKC group A: MGDG primarily stimulates PKC-α, and DAG primarily activates PKC-γ. The presence of these PKC isoforms in OLs was confirmed by western blotting, whereas PKC-β was only weakly stained, if at all. Addition of MGDG to the culture medium provided a higher density of regenerating OL fibers, which was not observed when membrane-permeable DAG was used. These findings indicate that MGDG can modulate the OL PKC activity and that PKC-α is the major PKC isoform involved in OL process formation.     

Protein kinase C in the human heart: differential regulation of the isoforms in aortic stenosis or dilated cardiomyopathy

Objectives Protein kinase C (PKC) is a central enzyme in the regulation of growth and hypertrophy. Little was known on PKC isoform regulation in human heart. Goal of this study was to characterize the isoforms of protein kinase C in human heart, their changes during ontogenesis, and their regulation in myocardial hypertrophy and heart failure. Methods In left ventricular and atrial samples from adults with end-stage dilated cardiomyopathy (DCM), from adults with severe aortic stenosis (AS), from small infants undergoing repair of ventricular septal defects, and from healthy organ donors (CO), activity of protein kinase C and the expression of its isozymes were examined. Results In the adult human heart, the isoforms PKC-α, PCK-β, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ were detected both on protein and on mRNA level. All isozymes are subjected to downregulation during ontogenesis. No evidence, however, exists for an isoform shift from infancy to adulthood. DCM leads to a pronounced upregulation of PKC-β. Severe left ventricular hypertrophy in AS, however, recruits a distinct isoform pattern, i.e., isoforms PKC-α, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ are upregulated, whereas PKC-β is not changed under this condition. Conclusion This work gives evidence for a differential recruitment of human PKC isoforms in various forms of myocardial hypertrophy and heart failure. Gregor Simonis and Steffen K. Briem contributed equally to this work.     

Staurosporine Induces Astrocytic Phenotypes and Differential Expression of Specific PKC Isoforms in C6 Glial Cells

Abstract: In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-β₂ and an increase in that of PKC-γ. In addition, it induced translocation of PKC-ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.     

Posttranslational Processing of Brain Actin

Abstract: Two short-lived isoforms of actin, named δ- and ε-actin, have been detected in brain extracts from rats labeled in vivo with [³⁵S]methionine. These two molecular species have PI values slightly more basic than β- and γ-actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ- and ε-actin, synthesized in vivo , can be converted in vitro into β- and γ-actin. This posttranslational processing of δ- and ε-actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH₂-terminal end, is involved in the transformation of these proteins into β- and γ-actin.     

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach 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 β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ 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.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach 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 β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ 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.