Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Photosystem II reaction centres stay intact during low temperature photoinhibition

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibition at low temperature by 1) creation of excessive excitation of Photosystem II or, 2) inhibition of the repair process of Photosystem II. The light and temperature dependence of the reduction state of Q_A was measured by modulated fluorescence. Photon flux densities giving 60% of Q_A in a reduced state at steady-state photosynthesis (300 mol m^–2s^–1 at 5°C and 1200 mol m^–2s^–1 at 20°C) resulted in a depression of the photochemical efficiency of Photosystem II (F_v/F_m) at both 5 and 20°C. Inhibition of F_v/F_m occurred with initially similar kinetics at the two temperatures. After 6h, F_v/F_m was inhibited by 30% and had reached steady-state at 20°C. However, at 5°C, F_v/F_m continued to decrease and after 10h, F_v/F_m was depressed to 55% of control. The light response of the reduction state of Q_A did not change during photoinhibition at 20°C, whereas after photoinhibition at 5°C, the proportion of closed reaction centres at a given photon flux density was 10–20% lower than before photoinhibition.Changes in the D1-content were measured by immunoblotting and by the atrazine binding capacity during photoinhibition at high and low temperatures, with and without the addition of chloramphenicol to block chloroplast encoded protein synthesis. At 20°C, there was a close correlation between the amount of D1-protein and the photochemical efficiency of photosystem II, both in the presence or in the absence of an active repair cycle. At 5°C, an accumulation of inactive reaction centres occurred, since the photochemical efficiency of Photosystem II was much more depressed than the loss of D1-protein. Furthermore, at 5°C the repair cycle was largely inhibited as concluded from the finding that blockage of chloroplast encoded protein synthesis did not enhance the susceptibility to photoinhibition at 5°C.It is concluded that, the kinetics of the initial decrease of F_v/F_m was determined by the reduction state of the primary electron acceptor Q_A, at both temperatures. However, the further suppression of F_v/F_m at 5°C after several hours of photoinhibition implies that the inhibited repair cycle started to have an effect in determining the photochemical efficiency of Photosystem II.Abbreviations CAP D-threochloramphenicol - F₀ and F ₀ fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively - F_m and F _m fluorescence when all Photosystem II reaction centres are closed in dark- and light-acclimated leaves, respectively - F_s fluorescence at steady state - Q_A the primary, stable quinone acceptor of Photosystem II - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence     

ABC50 Promotes Translation Initiation in Mammalian Cells

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.ABC50 was first reported as a protein whose expression is increased following treatment of synoviocytes with tumor necrosis factor α (1). ABC50 was subsequently identified independently as a protein that co-purified extensively with eukaryotic initiation factor 2 (eIF2)² (2). In common with other members of the ATP-binding cassette (ABC) family of proteins, ABC50 contains two ATP-binding cassettes (also termed nucleotide-binding domains (NBDs)) (1). Unlike most other members of the group, however, it lacks recognizable trans-membrane domains.Sequence analysis revealed that ABC50 is a close relative of the yeast protein Gcn20p, which is required for the control by amino acids of the yeast eIF2 kinase, Gcn2p, which is activated by binding to uncharged tRNA molecules (3). Gcn20p is thought to cooperate with Gcn1p to bring uncharged tRNAs to Gcn2p during the elongation process; this couples the availability of amino acids for tRNA charging to the control of Gcn2p (4). However, Gcn20p and ABC50 differ in important respects. For example, whereas Gcn20p associates with ribosomes that are engaged in elongation, ABC50 apparently binds ribosomes involved in initiation as well as elongation (2). Its association with ribosomes is stimulated by ATP. In addition, although Gcn20p and ABC50 are similar in their ABC domains, they differ markedly in their N termini. Since it is only the N terminus of Gcn20p that is required to support the function of Gcn2p in yeast (4), it seems likely that ABC50 and Gcn20p play distinct roles.Tyzack et al. (2) have provided initial data indicating that ABC50 stimulates the formation of complexes between eIF2, GTP, and the initiator methionyl-tRNA in vitro. It did so without affecting the binding of guanine nucleotides to eIF2, indicating that the effect is likely to be on the association of initiator methionyl-tRNA with eIF2. The available data thus suggested that ABC50 might play a positive role in the initiation of protein synthesis. However, no data for this have previously been presented. Similarly, the manner in which ABC50 binds to ribosomes, the significance of its ABC domains, and other features remained unclear.The two NBDs of ABC proteins are involved in nucleotide binding/hydrolysis and contain a number of conserved features, including the Walker box A and B motifs and the “ABC signature motif” (usually LSGGQ) (5, 6). The NBDs of eukaryotic ABC proteins “dimerize” such that the two ATP-binding/hydrolytic sites involve Walker box A of one NBD and the ABC signature motif of the other.Certain other non-membrane ABC proteins are known to be involved in translation or its control (7). Indeed, three of the eukaryotic ABCF classes contain proteins involved in the control of protein translation. Class I proteins are exemplified by ABC50 (also termed ABCF1). Class III proteins (exemplified by yeast Gcn20p) can interact with the ribosome in an ATP-dependent manner (4). The proteins of Class IVA (elongation factor 3) mediate translation elongation in certain fungi. eEF3 stimulates binding of the eEF1·GTP·aminoacyl-tRNA ternary complex to the ribosomal A site by facilitating the release of the deacylated tRNA from the E site, thus stimulating protein synthesis (8, 9). On the other hand, Class IVB contains proteins thought to be important for the export of mRNAs from the nucleus in yeast (10).The ABCE1 gene product was originally identified for its inhibition of ribonuclease L (11) and is hence also termed RLI1. Yeast Rli1p associates with 40 S ribosomal subunits in vivo and can interact with eIF3 and eIF5 independently of ribosomes (12). The available data indicate that ABCE1 is involved in both ribosome biogenesis and mRNA translation and shuttles between cytoplasm and nucleus, possibly as a nucleocytoplasmic transporter (13–17).Here, we report the first detailed investigation into the function and interactions of ABC50. The data described here identify features of ABC50 that are required for its interaction with ribosomes. Most importantly, we provide the first evidence that ABC50 is required for efficient translation initiation in living cells and show that the requirement for ABC50 differs between cap-dependent and internal ribosome entry segment (IRES)-dependent translation. These and other data indicate that the function of ABC50 is distinct from those of other ABC proteins.     

Mechanisms involved in the enhancement of mammalian target of rapamycin signalling and hypertrophy in skeletal muscle of myostatin-deficient mice

Myostatin deficiency leads to both an increased rate of protein synthesis and skeletal muscle hypertrophy. However, the mechanisms involved in mediating these effects are not yet fully understood. Here, we demonstrate that genetic loss of myostatin leads to enhanced muscle expression of both protein kinase B and mammalian target of rapamycin/S6K signalling components, consistent with their elevated activity. This is associated with a reduction in the expression of PGC1α and COX IV, proteins which play important roles in maintaining mitochondrial function. Furthermore, we show that these changes in signalling and protein expression are largely independent of alterations in intramuscular amino acid content. Our findings, therefore, reveal potential new mechanisms and further contribute to our understanding of myostatin-regulated skeletal muscle growth and function.     

Oligomycin sensitivity-conferring protein (OSCP) of beef heart mitochondria. Internal sequence homology and structural relationship with other proteins

Structural analysis of oligomycin sensitivity-conferring protein (OSCP) revealed repeating sequences (residues 1-89, 105-190) suggesting an evolution of the protein by gene duplication. In addition to the reported homology with the delta-subunit of Escherichia coli F1ATPase, OSCP also shows a certain homology with the b-subunit of E. coli F0 and the ADP/ATP carrier of mitochondria.     

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

Serotonin (5-Hydroxytryptamine), a novel regulator of glucose transport in rat skeletal muscle

In this study we show that serotonin (5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose uptake by approximately 50% in both L6 myotubes and isolated rat skeletal muscle. This activation is mediated via the 5-HT2A receptor, which is expressed in L6, rat, and human skeletal muscle. In L6 cells, expression of the 5-HT2A receptor is developmentally regulated based on the finding that receptor abundance increases by over 3-fold during differentiation from myoblasts to myotubes. Stimulation of the 5-HT2A receptor using methylserotonin (m-HT), a selective 5-HT2A agonist, increased muscle glucose uptake in a manner similar to that seen in response to 5-HT. The agonist-mediated stimulation in glucose uptake was attributable to an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4. The stimulatory effects of 5-HT and m-HT were suppressed in the presence of submicromolar concentrations of ketanserin (a selective 5-HT2A antagonist) providing further evidence that the increase in glucose uptake was specifically mediated via the 5-HT2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The present results indicate that rat and human skeletal muscle both express the 5-HT2A receptor and that 5-HT and specific 5-HT2A agonists can rapidly stimulate glucose uptake in skeletal muscle by a mechanism which does not depend upon components that participate in the insulin signaling pathway.     

Biochemical localisation of the 5-HT2A (serotonin) receptor in rat skeletal muscle

The 5-HT2A receptor was recently shown to localise morphologically to the transverse tubules (TT) in rat foetal myoblasts. Receptor activation enhanced the expression of genes involved in myogenesis, and its TT localisation has led to the suggestion that it may participate in excitation-contraction coupling. In order to gain further insights into 5-HT2A receptor function in muscle we have (i) investigated its biochemical localisation in adult rat skeletal muscle and (ii) determined whether receptor expression is dependent upon muscle type. Immunoblot analysis of muscle membranes, isolated by subcellular fractionation, revealed that adult muscle expresses the 5-HT2A receptor and that it resides exclusively in plasma membranes and not in TT. No differences in 5-HT2A abundance were observed between red and white muscle, suggesting that receptor expression does not correlate with the metabolic or contractile properties of the muscle fibre. Our data indicate that 5-HT2A expression in skeletal muscle is maintained into adulthood and that its absence from TT make it an unlikely participant in the excitation-contraction coupling process.     

Targeting of PKCzeta and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide

Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCzeta (protein kinase C-zeta), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCzeta-PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3'-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCzeta and PKB and suppressed kinase-caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1-/- mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.     

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids.

2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did not inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition.

3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols.

4. It is concluded that mitochondrial transhydrogenase, catalyzing the non-energy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.