To understand better the relative contributions of transcriptional and post-transcriptional processes towards the regulation of gene expression in plant mitochondria, we compared the steady state levels of RNAs with the respective transcriptional activities. All of the protein and rRNA coding genes of the Arabidopsis mitochondrial genome and several orfs were analyzed by run-on and northern experiments. rRNAs constitute the bulk of the steady state RNA in Arabidopsis mitochondria, but are (different from maize mitochondria) not equally prominent among the run-on transcripts. Their relatively low rate of active transcription is apparently compensated by their high stability. Run-on transcription values differ significantly between genes coding for different subunits of the same protein complex. The steady state RNA levels are considerably more homogeneous, indicating that high variations of transcription rates are counterbalanced by post-transcriptional processes. The relative amounts of the steady state transcripts for the different subunits in a given protein complex reflect the relative stoichiometries of the protein subunits much more closely than the respective transcriptional activities. Posttranscriptional RNA processing and stability thus contribute significantly to the regulation of gene expression in Arabidopsis mitochondria.

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G␣ subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G␣ subunits of heterotrimeric G-proteins. Parathormone (PTH)1 secretion from the parathyroid gland is suppressed by high extracellular calcium and magnesium (1). The calcium-sensing receptor (CaSR) is responsible for the calcium-dependent inhibition of PTH secretion (2). Direct binding of calcium or magnesium activates the CaSR (3). Activation of the CaSR triggers G␣ q /G␣ i -mediated signaling pathways (4). Several mutations have been identified with increased activation of this receptor (5, 6). CaSR mutants with increased affinity/potency for the agonist calcium and in part enhanced constitutive activity led to permanent inhibition of PTH secretion (7). Therefore, patients with activated CaSR mutants suffer from hypoparathyroidism. A similar phenotype of blunted PTH secretion is seen in patients with severe magnesium deficiency (8 -10). This finding is unexpected since the effects of high magnesium on parathyroid hormone secretion are similar to those of calcium, and therefore, low magnesium should be expected to result in increased PTH secretion. And indeed, in contrast to patients, rats respond to severe hypomagnesemia with increased secretion of PTH (11,12). It is known that hypomagnesemia reflects intracellular magnesium deficiency (9). Thus, the site of magnesium action has been assumed to lie intracellularly (9). However, causality between blunted PTH secretion and magnesium deficiency is not established, although the magnesium paradox has been known for more than 20 years (8). In search for the mechanism we investigated the relationship between magnesium deficiency, PTH secretion, and CaSR-mediated signaling...

G-protein-coupled receptor kinase 2 (GRK2) is activated by free G␤␥ subunits. A G␤␥ binding site of GRK2is localized in the carboxyl-terminal pleckstrin homology domain. This G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits. We report here that truncation of the carboxyl-terminal G␤␥ binding site of GRK2 did not abolish the G␤␥ regulatory activity of GRK2 as determined by the inhibition of a G␤␥-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second G␤␥ binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2 1-185 ) inhibited a G␤␥-stimulated inositol phosphate signal in cells, purified GRK2 1-185 suppressed the G␤␥-stimulated phosphorylation of rhodopsin, and GRK2 1-185 bound directly to purified G␤␥ subunits. The amino-terminal G␤␥ regulatory site does not overlap with the RGS domain of GRK-2 because GRK2 1-53 with truncated RGS domain inhibited G␤␥-mediated signaling with similar potency and efficacy as did GRK2 1-185 . In addition to the G␤␥ regulatory activity, the amino-terminal G␤␥ 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 G␤␥ subunits, strongly suggesting that G␤␥ binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized G␤␥ binding site that regulates GRK2-mediated receptor phosphorylation and inhibits G␤␥-stimulated signaling.Activated G-protein-coupled receptors are switched off by phosphorylation through G-protein-coupled receptor kinases (GRKs) 1 (1). GRKs are modular proteins consisting of at least three structural domains with different functions. The core kinase domain of GRK2 and GRK3, which represents the ␤-adrenergic receptor kinase isozymes, is flanked by an aminoterminal domain, which contains an RGS domain, and a carboxyl-terminal domain, which contains a pleckstrin homology domain (PH domain) (2-4). The activation of GRK2 and GRK3 requires the activation and dissociation of a heterotrimeric G-protein, i.e. the kinases are activated by free G␤␥ subunits (5, 6). A G␤␥ binding site of GRK2 and GRK3 is localized in the carboxyl terminus of the kinase and overlaps the PH domain (7). Truncation of the PH domain of GRK2 generates a kinase with compromised regulation by G␤␥ subunits (7). The carboxyl-terminal G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits (8).Analyzing the G␤␥ regulatory activity of proteins is a means of identifying G␤␥-binding proteins or localizing G␤␥ binding sites of proteins (9 -11). To find out whether the G␤␥ regulatory activity of GRK2 resides entirely in the carboxyl-terminal PH domain, we analyzed the G␤␥-sequestering activity of wildtype GRK2 and of carboxyl-terminal-truncated GRK2 mutants. The capacity of thos...

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. Tetrahydrofolate (THF)5 and its one-carbon-substituted derivatives (collectively termed folates) are involved in key metabolic functions, including the synthesis of methionine, pantothenate, purines, and thymidylate (1). Plants and most microorganisms can synthesize THF de novo, whereas mammals cannot and so require a dietary supply of this soluble vitamin. The plant THF biosynthesis has a unique and complex subcellular compartmentation split between three compartments (Fig.