scholarly journals Subcellular structure containing mRNA for β subunit of mitochondrial H+-ATP synthase in rat hepatocytes is translationally active

1997 ◽  
Vol 324 (2) ◽  
pp. 635-643 ◽  
Author(s):  
Javier RICART ◽  
Gustavo EGEA ◽  
José M. IZQUIERDO ◽  
SAN MARTÍN Carmen ◽  
José M. CUEZVA
Keyword(s):  
Atp Synthase ◽  
Rat Hepatocytes ◽  
Translational Efficiency ◽  
Β Subunit ◽  
F1 Atpase ◽  
Close Proximity ◽  
Show Evidence ◽  
Positive Hybridization ◽  
Cytoplasmic Location

We have recently reported that the nuclear-encoded mRNA for the β subunit of mitochondrial H+-ATP synthase (β-mRNA) is localized in rounded, electron-dense clusters in the cytoplasm of rat hepatocytes. Clusters of β-mRNA are often found in close proximity to mitochondria. These findings suggested a role for these structures in controlling the cytoplasmic expression and sorting of the encoded mitochondrial precursor. Here we have addressed the question of whether the structures containing β-mRNA are translationally active. For this purpose a combination of high-resolution in situ hybridization and immunocytochemical procedures was used. Three different co-localization criteria showed that β-mRNA-containing structures always revealed positive immunoreactive signals for mitochondrial H+-ATP synthase (F1-ATPase), ribosomal and hsc70 proteins. Furthermore, clusters show evidence in situ of developmental changes in the translational efficiency of the β-mRNA. These findings suggest that structures containing β-mRNA are translationally active irrespective of their cytoplasmic location. The immunocytochemical quantification of the cytoplasmic presentation of hsc70 in the hepatocyte reveals that approx. 86% of the protein has a dispersed distribution pattern. However, the remaining hsc70 is presented in clusters of which only half reveal positive hybridization for β-mRNA. The interaction of hsc70 with the β-F1-ATPase precursor protein is documented by the co-localization of F1-ATPase immunoreactive material within cytoplasmic clusters of hsc70 and by the co-immunoprecipitation of hsc70 with the β-subunit precursor from liver post-mitochondrial supernatants. Taken together, these results suggest a role for hsc70 in the translation/sorting pathway of the mammalian precursor of the β-F1-ATPase protein.

scholarly journals mRNA encoding the β-subunit of the mitochondrial F1-ATPase complex is a localized mRNA in rat hepatocytes

1997 ◽  
Vol 322 (2) ◽  
pp. 557-565 ◽  
Author(s):  
Gustavo EGEA ◽  
José M. IZQUIERDO ◽  
Javier RICART ◽  
Carmen SAN MARTÍN ◽  
José M. CUEZVA
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Rat Hepatocytes ◽  
Mrna Localization ◽  
Β Subunit ◽  
Control Of Gene Expression ◽  
F1 Atpase ◽  
Mrna Species ◽  
Close Proximity

Subcellular mRNA localization has emerged as a mechanism for regulation of gene expression and protein-sorting pathways. Here we describe the different cytoplasmic presentation in rat hepatocytes of two nuclear mRNA species encoding subunits α and β of the mitochondrial F1-ATPase complex. α-F1-ATPase mRNA is dispersed and scattered in the cytoplasm. In contrast, β-F1-ATPase mRNA appears in rounded electron-dense clusters, often in close proximity to mitochondria. Hybridization experiments with β2-microglobulin and β-actin cDNA species reveal an expected subcellular distribution pattern of the mRNA species and a non-clustered appearance. Development does not alter the presentation of β-F1-ATPase mRNA hybrids, although it affects the relative abundance of β-F1-ATPase mRNA clusters in the cytoplasm of the hepatocyte. These findings illustrate in vivo the existence of two different sorting pathways for the nuclear-encoded mRNA species of mitochondrial proteins. High-resolution immunocytochemistry and immunoprecipitation experiments allowed the identification of the β-subunit precursor in the cytoplasm of the hepatocyte, also suggesting a post-translational import pathway for this precursor protein. It is suggested that the localization of β-F1-ATPase mRNA in a subcellular structure of the hepatocyte might have implications for the control of gene expression at post-transcriptional levels during mitochondrial biogenesis in mammals.

scholarly journals Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

2005 ◽  
Vol 386 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Hamid R. SAMARI ◽  
Michael T. N. MØLLER ◽  
Lise HOLDEN ◽  
Tonje ASMYHR ◽  
Per O. SEGLEN
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Calyculin A ◽  
Β Subunit ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

scholarly journals Assembly of the ribonucleoprotein complex containing the mRNA of the β-subunit of the mitochondrial H+-ATP synthase requires the participation of two distal cis-acting elements and a complex set of cellular trans-acting proteins

2002 ◽  
Vol 365 (2) ◽  
pp. 417-428 ◽  
Author(s):  
Javier RICART ◽  
José M. IZQUIERDO ◽  
Carlo M. Di LIEGRO ◽  
José M. CUEZVA
Keyword(s):  
Atp Synthase ◽  
Sucrose Concentration ◽  
Β Subunit ◽  
Reading Frame ◽  
Ribonucleoprotein Complex ◽  
Cis Acting ◽  
Translational Machinery ◽  
F1 Atpase ◽  
Reticulocyte Lysate

The mRNA encoding the β-subunit of the mitochondrial H+-ATP synthase (β-F1-ATPase) is localized in an approx. 150nm structure of the hepatocyte of mammals. In the present study, we have investigated the cis- and trans-acting factors involved in the generation of the ribonucleoprotein complex containing β-F1-ATPase mRNA. Two cis-acting elements (β1.2 and 3′β) have been identified. The β1.2 element is placed in the open reading frame, downstream of the region encoding the mitochondrial pre-sequence of the protein. The 3′β element is the 3′ non-translated region of the mRNA. Complex sets of proteins from the soluble and non-soluble fractions of the liver interact with the β1.2 and 3′β elements. A soluble p88, present also in reticulocyte lysate, displays binding specificity for both the cis-acting elements. Sedimentation and high-resolution in situ hybridization experiments showed that the structure containing the rat liver β-F1-ATPase mRNA is found in fractions of high sucrose concentration, where large polysomes sediment. Treatment of liver extracts with EDTA promoted the mobilization of β-F1-ATPase mRNA to fractions of lower sucrose concentration, suggesting that the structure containing β-F1-ATPase mRNA is a large polysome. Finally, in vitro reconstitution experiments with reticulocyte lysate, using either the full-length, mutant or chimaeric versions of β-F1-ATPase mRNA, reveal that the assembly of the β-F1-ATPase mRNA polysome requires the co-operation of both the cis-acting mRNA determinants. The present study illustrates the existence of an intramolecular RNA cross-talking required for the association of the mRNA with the translational machinery.

scholarly journals The Mitochondrial Genome Integrity Gene, MGI1, of Kluyveromyces lactis Encodes the β-Subunit of F1-ATPase

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1445-1454 ◽  
Author(s):  
Xin Jie Chen ◽  
G Desmond Clark-Walker
Keyword(s):  
Mitochondrial Genome ◽  
Kluyveromyces Lactis ◽  
Three Dimensional ◽  
Genome Integrity ◽  
Dimensional Structure ◽  
Deletion Mutants ◽  
Β Subunit ◽  
Gain Of Function ◽  
F1 Atpase ◽  
Γ Subunit

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

1996 ◽  
Vol 154 (2) ◽  
pp. 107-111 ◽  
Author(s):  
Immaculada Martin ◽  
Josep A. Villena ◽  
Marta Giralt ◽  
Roser Iglesias ◽  
Teresa Mampel ◽  
...  
Keyword(s):  
Thyroid Hormones ◽  
Atp Synthase ◽  
Gene Promoter ◽  
Β Subunit ◽  
Subunit Gene

scholarly journals Specificity of Associations between Bacteria and the Coral Pocillopora meandrina during Early Development

2012 ◽  
Vol 78 (20) ◽  
pp. 7467-7475 ◽  
Author(s):  
Amy Apprill ◽  
Heather Q. Marlow ◽  
Mark Q. Martindale ◽  
Michael S. Rappé
Keyword(s):  
Small Subunit ◽  
Rrna Genes ◽  
Polymorphism Analysis ◽  
Roseobacter Clade ◽  
Small Subunit Rrna ◽  
Content Type ◽  
Sterile Seawater ◽  
Show Evidence ◽  
Coral Health

ABSTRACTRelationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coralPocillopora meandrinawas investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of theAlphaproteobacteria, aPseudoalteromonasspecies of theGammaproteobacteria, and aSynechococcusspecies of theCyanobacteriaphylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination ofP. meandrinaplanulae by fluorescencein situhybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains ofPseudoalteromonasand Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade andSynechococcusdid not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.

scholarly journals Biochemical and Genetic Analysis of the Mitochondrial Response of Yeast to BAX and BCL-XL

2000 ◽  
Vol 20 (9) ◽  
pp. 3125-3136 ◽  
Author(s):  
Atan Gross ◽  
Kirsten Pilcher ◽  
Elizabeth Blachly-Dyson ◽  
Emy Basso ◽  
Jennifer Jockel ◽  
...  
Keyword(s):  
Cell Death ◽  
Mitochondrial Genome ◽  
Atp Synthase ◽  
Mitochondrial Permeability Transition ◽  
Family Members ◽  
Growth Arrest ◽  
Anion Channel ◽  
Cell Killing ◽  
Β Subunit ◽  
Voltage Dependent Anion Channel

ABSTRACT The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-XL) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-XL using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-XL prevented all BAX-mediated responses. We also assessed the function of BCL-XL and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic β subunit or the δ subunit of the F0F1-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho 0] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the β subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-XL protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-XL: cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.

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