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MRNA encoding the beta-subunit of the mitochondrial F1-ATPase complex is a localized mRNA in rat hepatocytes.

ABSTRACT: 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 alpha and beta of the mitochondrial F1-ATPase complex. alpha-F1-ATPase mRNA is dispersed and scattered in the cytoplasm. In contrast, beta-F1-ATPase mRNA appears in rounded electron-dense clusters, often in close proximity to mitochondria. Hybridization experiments with beta2-microglobulin and beta-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 beta-F1-ATPase mRNA hybrids, although it affects the relative abundance of beta-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 beta-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 beta-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.

SUBMITTER: Egea G

PROVIDER: S-EPMC1218226 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Conformational dynamics of the F1-ATPase beta-subunit: a molecular dynamics study.

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| S-EPMC1303325 | biostudies-literature

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Project description:ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.

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Deciphering Intrinsic Inter-subunit Couplings that Lead to Sequential Hydrolysis of F1-ATPase Ring.

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