Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Animal and plant mitochondria contain specific thioredoxins

Thioredoxins have been purified from pig heart and potato tuber mitochondria which differ in chromatographic behaviour, enzyme activating capacity, and slightly higher molecular mass (Mr = 12,500) from the major thioredoxin(s) present in mitochondria-free fractions of the same tissue. Both mt-thioredoxins can serve as hydrogen donor for E. coli ribonucleotide reductase but only the plant protein activates spinach chloroplast NADP malate dehydrogenase in vitro. Mitochondrial target enzymes specifically activated by thioredoxin have not as yet been identified.     

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.     

Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells

Yeast cells permeabilized by freeze-thaw cycles in a sorbitol-containing medium provide an experimentally favorable system for the study of ribonucleotide reduction in a small number of cells or in mutant strains. Ribonucleotide reductase activities determined in such cells are about twice those found in cell extracts but properties of the enzyme, except pH optimum, are closely comparable in both assay procedures. In contrast with other organisms, the activities measured in permeabilized cells from both diploid or haploid strains exceed the demand for deoxyribonucleotide formation during replication of the yeast genome. The method has been applied to yeast cultures growing in the presence of the ribonucleotide reductase inhibitor hydroxyurea and a twofold increase of enzyme activity has been established in such cells. On the other hand, analysis of a series of hus mutants, selected for hydroxyurea sensitivity in the laboratory of Singer and Johnston did not reveal obvious alterations of the enzyme vs the parental strains, suggesting that the hus phenotype may be due to lesions other than in ribonucleotide reductase.     

Deoxyribonucleotide biosynthesis in green algae: characterization of thymidylate synthase-dihydrofolate reductase in Scenedesmus obliquus

Thymidylate synthase and dihydrofolate reductase are peak enzymes that accompany the S phase of the unicellular green algae, Scenedesmus obliquus, and are both overproduced in the presence of 5-fluorodeoxyuridine. Such overproducing cultures have served for enzyme isolation and characterization. It has not been possible to separate the two enzyme activities by several methods of protein fractionation, including affinity chromatography on specific immobilized ligands (fluorodeoxyuridylate or N10-formylfolate); both were enriched in parallel approximately 400-fold from algal extracts. The most highly purified samples are of low stability in solution. Enzyme activities are inhibited by methotrexate, 5-fluorodeoxyuridylate, and arabinouridylate but not by hydroxyurea; FdUMP inhibition is fully reversed after removal of the nucleotide. Sedimentation in sucrose gradients (Mr 100,000) and electrophoresis in denaturing polyacrylamide gels (Mr 50,000) suggest that the protein structure resembles more the dimeric, bifunctional thymidylate synthase-dihydrofolate reductase of protozoan species than the separate enzymes found in bacteria and animal cells.     

Contrasted levels of genetic diversity in a benthic Mediterranean octocoral: Consequences of different demographic histories?

Understanding the factors explaining the observed patterns of genetic diversity is an important question in evolutionary biology. We provide the first data on the genetic structure of a Mediterranean octocoral, the yellow gorgonian Eunicella cavolini, along with insights into the demographic history of this species. We sampled populations in four areas of the Mediterranean Sea: continental France, Algeria, Turkey, and the Balearic and Corsica islands. Along French coasts, three sites were sampled at two depths (20 and 40 m). We demonstrated a high genetic structure in this species (overall F_ST = 0.13), and most pairwise differentiation tests were significant. We did not detect any difference between depths at the same site. Clustering analyses revealed four differentiated groups corresponding to the main geographical areas. The levels of allelic richness and heterozygosity were significantly different between regions, with highest diversity in Algeria and lowest levels in Turkey. The highest levels of private allelic richness were observed in Algeria followed by Turkey. Such contrasted patterns of genetic diversity were not observed in other Mediterranean octocorals and could be the result of different evolutionary histories. We also provide new empirical evidence of contrasting results between tests and model‐based studies of demographic history. Our results have important consequences for the management of this species.     

Short-term effects of wildfire on microbial biomass and abundance in black pine plantation soils in Turkey

Measurement of soil microbial biomass and abundance offers a means of assessing the response of all microbial populations to changes in the soil environment after a fire. We examined the effects of wildfire on microbial biomass C and N, and abundance of bacteria and fungi 2 months after a fire in a pine plantation. Soil organic carbon (C_org), total nitrogen (N_tot), and electrical conductivity (EC) increased following the fire. In terms of microbial abundance, the overall results showed that burned forest soils had the most bacteria and fungi. Microbial biomass C and N from soil in the burned forest were not significantly different from their unburned forest counterparts. However, microbial indices indicated that fire affects soil microbial community structure by modifying the environmental conditions. The results also suggested that low-intensity fire promotes microorganism functional activity and improves the chemical characteristics of soils under humid climatic conditions.