Nigel T. Price

Lactic Acid Efflux from White Skeletal Muscle Is Catalyzed by the Monocarboxylate Transporter Isoform MCT3

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40–60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2–3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3‐kinase (PI 3‐kinase). The ϵ‐subunit of eIF2B is phosphorylated by glycogen synthase kinase‐3 (GSK‐3), which is inactivated by insulin in a PI 3‐kinase‐dependent manner. Here we identify the phosphorylation site in eIF2Bϵ as Ser540 and show that treatment of eIF2B with GSK‐3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3‐kinase inhibitors. Insulin‐induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

Lactate transport in heart in relation to myocardial ischemia

In this article, the importance of lactic acid transport into and out of heart cells is described and the properties of the monocarboxylate transporters (MCTs) responsible are presented. These are monocarboxylate/proton symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. Although it is unlikely that lactic acid transport constrains heart metabolism under most conditions, it may do so during severe hypoxia or ischemia. The transporter plays a critical role in maintaining intracellular pH because it removes the protons that are produced stoichiometrically with lactate during glycolysis. The kinetics and substrate and inhibitor specificities of the transport process have been determined in cell suspensions using a radiotracer technique and in single cells using a fluorescent measurement of the decrease in intracellular pH that accompanies transport. The results of these experiments suggest the presence of 2 different transporter isoforms in heart cells, at least one of which is different from the cloned MCT1 and MCT2. Immunofluorescence microscopy shows that MCT1 expression is restricted to the intercalated disk region, yet the rate of lactate transport in this region is slower than in the center of the cell, where there is no MCT1. New cDNA sequences with strong homology to MCT1 have been found in human cDNA libraries and Northern blots show that the corresponding mRNA is expressed in rat heart. Expressions of these new MCT isoforms have yet to be demonstrated and their properties and cellular distribution defined.

The guanine nucleotide-exchange factor, eIF-2B

Eukaryotic initiation factor eIF-2B catalyses the exchange of guanine nucleotides on another translation initiation factor, eIF-2, which itself mediates the binding of the initiator Met-tRNA to the 40S ribosomal subunit during translation initiation. eIF-2B promotes the release of GDP from inactive [eIF-2.GDP] complexes, thus allowing formation of the active [eIF-2.GTP] species which subsequently binds the Met-tRNA. This guanine nucleotide-exchange step, and thus eIF-2B activity, are known to be an important control point for translation initiation. The activity of eIF-2B can be modulated in several ways. The best characterised of these involves the phosphorylation of the alpha-subunit of eIF-2 by specific protein kinases regulated by particular ligands. Phosphorylation of eIF-2 alpha leads to inhibition of eIF-2B. This mechanism is involved in the control of translation under a variety of conditions, including amino acid deprivation in yeast (Saccharomyces cerevisiae) where it causes translational upregulation of the transcription factor GCN4, and in virus-infected animal cells, where it involves a protein kinase activated by double-stranded RNA. There is now also growing evidence for direct regulation of eIF-2B. This appears likely to involve the phosphorylation of its largest subunit. Under certain circumstances eIF-2B may also be regulated by allosteric mechanisms. eIF-2B is a heteropentamer (subunits termed alpha, beta, gamma, delta and epsilon) and is thus more complex than most other guanine nucleotide-exchange factors. The genes encoding all five subunits have been cloned in yeast (exploiting the GCN4 regulatory system): all but the alpha appear to be essential for eIF-2B activity. However, this subunit may confer sensitivity to eIF-2 alpha phosphorylation. cDNAs encoding the alpha, beta, delta and epsilon subunits have been cloned from mammalian sources. There is substantial homology between the yeast and mammalian sequences. Attention is now directed towards understanding the roles of individual subunits in the function and regulation of eIF-2B.

Identification of the phosphorylation sites in elongation factor-2 from rabbit reticulocytes

The sites in eukaryotic elongation factor eEF‐2 phosphorylated by the Ca2+/calmodulin‐dependent eEF‐2 kinase in vitro have been identified. The kinase catalysed the rapid incorporation of one mol of phosphate per mol eEF‐2 and the slower incorporation of a second mol. All the phosphorylation sites in eEF‐2 are contained in the CNBr fragment corresponding to residues 22‐155. Tryptic digestion of phosphorylated eEF‐2 yielded 3 phosphopeptides, one being unique to monophosphorylated eEF‐2. The phosphorylation sites were identified as threonine residues 56 and 58, the former being more rapidly phosphorylated. Ala‐Gly‐Glu‐Thr‐Phe‐Thr14‐Asp‐Thr18‐Arg. The same sites are labelled in eEF‐2 isolated from reticulocyte lysates.

cDNA cloning of rat mitochondrial cyclophilin

Human cyclophilin-3 (hCyp-3) cDNA was used to screen a rat skeletal muscle cDNA library which led to the isolation of a full-length cDNA encoding the rat homologue. The derived amino acid sequence showed 89.5% identity with the human sequence, with major differences being restricted to the mitochondrial targeting sequence. The N-terminal sequence of the purified rat liver mitochondrial cyclophilin (CyP-D) corresponded to that derived from the cDNA following 30 amino acids of targeting sequence. This confirms that hCyp-3 encodes mitochondrial matrix CyP (CyP-D), which plays a crucial role in the mitochondrial permeability transition. CyP-D mRNA of a single sized (1.5 kb) was shown by Northern blotting to be present in liver, heart, skeletal muscle and brain.

CDNA CLONING OF MCT1, A MONOCARBOXYLATE TRANSPORTER FROM RAT SKELETAL MUSCLE

PCR was used to amplify the coding region of CHO MCT1 cDNA. This was then used to screen a rat skeletal muscle cDNA library which lead to the isolation of a full length cDNA encoding MCT1 from rat. The cDNA derived amino acid sequence shows 94% and 86% identity to CHO and human MCT1, respectively.

Cloning and Expression of cDNA Encoding Protein Synthesis Elongation Factor-2 Kinase

A cDNA from rat skeletal muscle encoding calcium/calmodulin-dependent eukaryotic elongation factor-2 kinase (eEF-2K) has been cloned and sequenced, and the amino acid sequence of the protein has been deduced. The kinase is composed of 724 amino acids and has a predicted molecular mass of 81,499 Da. The cDNA was judged to be full-length, as the protein, expressed in rabbit reticulocyte lysate or wheat germ extract, migrated upon SDS-PAGE with the same apparent molecular weight as the purified kinase and possessed eEF-2K activity. eEF-2K contains all of the 12 catalytic subdomains present in the majority of protein kinases, but they are atypical and display only limited homology with other kinases. A putative calmodulin-binding domain is present C-terminal to the catalytic domain as is a putative pseudosubstrate sequence. Two antipeptide antibodies raised against sequences derived from a partial rabbit cDNA clone, cross-reacted with purified eEF-2K, and one also immunoprecipitated eEF-2K activity from cell extracts. Northern blot analysis demonstrated that eEF-2K mRNA is expressed in a number of different tissues and that it may exist in multiple forms.

Characterization of the Mammalian Initiation Factor eIF2B Complex as a GDP Dissociation Stimulator Protein

Initiation factor eIF2B mediates a key regulatory step in the initiation of mRNA translation, i.e. the regeneration of active eIF2·GTP complexes. It is composed of five subunits, α–ε. The largest of these (ε) displays catalytic activity in the absence of the others. The catalytic mechanism of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammalian eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2·eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2Bε itself bind GTP. However, we show that it is the β-subunit of eIF2B that interacts with GTP. This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparations that lack the α-subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2Bα markedly enhances activity, indicating that eIF2Bα is required for full activity of mammalian eIF2B.

The two forms of the β-subunit of initiation factor-2 from reticulocyte lysates arise from proteolytic degradation

Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the beta-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed beta H (heavy, the slower-migrating species, apparent Mr = 54,300) and beta L (light, the faster-migrating species, apparent Mr = 53,100). We confirm that two forms of eIF-2 beta are present in such preparations, but present evidence that the beta L is generated from beta H during the isolation procedure. Crude reticulocyte lysates contain only the beta H species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2 beta. The beta L species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the beta H and the beta L forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2 beta, the beta L form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the beta-subunit.