Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes.DOI: http://dx.doi.org/10.7554/eLife.03640.001

Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. The underlying mechanism is unknown. Previous studies have shown that tacrolimus can inhibit insulin gene transcription at high concentrations in tumor cell lines. To study insulin gene transcription in normal, mature pancreatic islet cells, we used a novel approach in the present study. Transgenic mice that carry a human insulin promoterreporter gene were generated. The human insulin promoter directed transcription in pancreatic islets and conferred a normal, physiological glucose response to reporter gene expression in isolated islets. After stimulation with glucose, human insulin promoter-mediated gene expression was inhibited in normal, mature islet cells by both tacrolimus and cyclosporin A to a large extent (approximately 70%) and with high potency at concentrations that are known to inhibit calcineurin phosphatase activity (IC 50 values of 1 and 35 nM, respectively). Furthermore, glucose stimulated calcineurin phosphatase activity in mouse pancreatic islets, further supporting the view that calcineurin phosphatase activity is an essential part of glucose signaling to the human insulin gene. The high potency of cyclosporin A and tacrolimus in normal islets suggests that inhibition of insulin gene transcription by cyclosporin A and tacrolimus is clinically important and is one mechanism of the diabetogenic effect of these immunosuppressive drugs.Cyclosporin A and tacrolimus (also known as FK506) are powerful and clinically important immunosuppressive drugs that are widely used to prevent organ rejection after transplantation. In addition, an increasing number of autoimmune diseases are treated with these drugs. Both structurally distinct drugs bind to their respective intracellular receptors, the immunophilins, and the drug-immunophilin complexes then bind to and inhibit calcineurin phosphatase (Ho et al., 1996). Inhibition of calcineurin prevents the dephosphorylation of the nuclear factor of activated T cells (NFAT) and its nuclear translocation, resulting in decreased transcription of NFAT-dependent genes. Additional targets of calcineurin, such as the transcription factor cAMP response element-binding protein CREB (Schwaninger et al., 1993(Schwaninger et al., , 1995Barton et al., 1996;Krü ger et al., 1997), are likely to be involved. In this way, cyclosporin A and tacrolimus block early steps in T-cell activation.Among the most serious adverse effects of cyclosporin A and tacrolimus is an impaired glucose tolerance leading to hyperglycemia and diabetes mellitus (Kahan, 1989;Docherty and Clark, 1994;European FK506 Multicentre Liver Study

1 In insulin-secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH 7.4 compound IV is only present in charged form. 2 Lipid solubility declined in the order tolbutamide > compound III > compound IV. 3 The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT-cells (pancreatic P-cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4 In mouse pancreatic P-cells, drug concentrations inhibiting adenosine 5'-triphosphate-sensitive K+ channels (KATP-channels) half-maximally (EC5() were determined by use of the patch-clamp technique.When the drugs were applied to the extracellular side of outside-out or the intracellular side of inside-out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide. 5In mouse pancreatic P-cells, measurement of KATp-channel activity in cell-attached patches and recording of insulin release displayed much higher EC.% values for compound IV than inside-out patch experiments. A corresponding, but less pronounced difference in EC.V values was observed for compound III, whereas the ECv values for tolbutamide did not differ significantly.6 It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the P-cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.

1 In mouse pancreatic P-cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5'-triphosphate-sensitive K+ channel (KATP-channel) was examined. Patchclamp experiments with excised inside-out membrane patches were carried out using an experimental protocol favouring phosphorylation of membrane proteins.2 In the absence of Mg2+, the KATP-channel-inhibiting potency of cytosolic nucleotides decreased inIn the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATPyS > ATP > AMP-PNP > ADPPS > dATP > UTP. In the presence of Mg2", the KATp-channels were activated by dADP, GTP, GDP and UDP.4 Tolbutamide inhibited the KATp-channels not only in the presence but also in the prolonged absence of Mg2+. In nucleotide-free solutions, the potency of tolbutamide was very low. When about half of the KATp-channel activity was inhibited by ATP, AMP-PNP, ADPPS or ADP (absence of Mg2+), the potency of tolbutamide was increased.5 Tolbutamide (100 IM) slightly enhanced the channel-inhibiting potency of AMP-PNP and inhibited the channel-activating effect of MgGDP in a non-competitive manner. 6 Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP-PNP (1 ILM-1 mM). This effect of GDP was neutralized by tolbutamide (100 M). 7 The stimulatory effect of 0.5 mM MgGDP was neutralized by 200 11M AMP-PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the absence of any nucleotides.8 dADP (0.3-1 mM) increased the potency of tolbutamide. Additional application of 200 f1M AMP-PNP caused a further increase in the potency of tolbutamide. 9 In conclusion, in the simultaneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel activity, non-competitive inhibition of the action of stimulatory nucleotides and interruption of the competitive interaction between stimulatory and inhibitory nucleotides. The latter effect increases the proportion of KATP-channels staying in the nucleotide-blocked state. In addition, this state potentiates the direct effect of sulphonylureas.

1. In mouse pancreatic P-cells the regulation of the diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel (K-ATP-channel) was 5. Diazoxide (50 or 300 jM) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2" and ADP (1 mM). 6. In the presence of Mg2", the K-ATP-channel-blocking potency of cytosolic ATP decreased in the order inside-out> outside-out> whole-cell configuration of the patch-clamp technique. 7. It is concluded that the K-ATP-channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.