RESUMEN
The distribution of folates in plant cells suggests a complex traffic of the vitamin between the organelles and the cytosol. The Arabidopsis thaliana protein AtFOLT1 encoded by the At5g66380 gene is the closest homolog of the mitochondrial folate transporters (MFTs) characterized in mammalian cells. AtFOLT1 belongs to the mitochondrial carrier family, but GFP-tagging experiments and Western blot analyses indicated that it is targeted to the envelope of chloroplasts. By using the glycine auxotroph Chinese hamster ovary glyB cell line, which lacks a functional MFT and is deficient in folates transport into mitochondria, we showed by complementation that AtFOLT1 functions as a folate transporter in a hamster background. Indeed, stable transfectants bearing the AtFOLT1 cDNA have enhanced levels of folates in mitochondria and can support growth in glycine-free medium. Also, the expression of AtFOLT1 in Escherichia coli allows bacterial cells to uptake exogenous folate. Disruption of the AtFOLT1 gene in Arabidopsis does not lead to phenotypic alterations in folate-sufficient or folate-deficient plants. Also, the atfolt1 null mutant contains wild-type levels of folates in chloroplasts and preserves the enzymatic capacity to catalyze folate-dependent reactions in this subcellular compartment. These findings suggest strongly that, despite many common features shared by chloroplasts and mitochondria from mammals regarding folate metabolism, the folate import mechanisms in these organelles are not equivalent: folate uptake by mammalian mitochondria is mediated by a unique transporter, whereas there are alternative routes for folate import into chloroplasts.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas de Transporte de Membrana/química , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/fisiología , Western Blotting , Células CHO , Catálisis , Clorofila/química , Cloroplastos/química , Clonación Molecular , Cricetinae , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Prueba de Complementación Genética , Glicina/química , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Mitocondrias/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Ácidos Nucleicos/química , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , TransfecciónRESUMEN
G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Sitios de Unión , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Quinasas de Receptores Adrenérgicos betaRESUMEN
Sequences ranging from nucleotide positions -14 to +4 relative to the transcription start site constitute an in vitro functional pea atp9 promoter. A comparison of respective sequence segments surrounding 11 unambiguously identified transcription initiation sites of various dicotyledoneous plant species revealed the highest level of evolutionary fidelity of nucleotide identities within the conserved nonanucleotide motif (CNM), suggesting their importance for promoter function. Using a mitochondrial in vitro transcription system, a detailed analysis by site-directed mutagenesis now reveals that the alteration of nucleotides -6 to -2 and +1 within the CNM indeed reduces promoter activity by more than 80%. Changes of nucleotide identities at the less conserved positions -12 to -9 within the AT-rich region reduced the initiation efficiency by about 70%. The alteration of the highly conserved position -7 has little influence on promoter function, indicating that evolutionary conservation does not always correlate with the functional importance of certain nucleotides. Mutagenesis of nucleotides at positions +3 or +4 reveals a minimal requirement of at least one purine for wild-type transcription initiation efficiency. The assignment of functionally important nucleotide identities should now facilitate an efficient and reliable prediction of other promoters in mitochondria of dicotyledon plants.