Putative Translocator Research Articles

Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator.

We have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1,179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described.

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. haliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

Identification of the putative hexose-phosphate translocator of amyloplasts from cauliflower buds

Starch synthesis in amyloplasts isolated from cauliflower buds is strongly inhibited by the addition of micromolar concentrations of 4,4'-di-isothiocyano-2,2'-stilbenedisulphonic acid (DIDS). Using [3H]DIDS it was possible to label specifically a 31.6 kDa membrane protein of the envelope fraction of isolated amyloplasts. The intensity of the radioactive label was decreased in the presence of glucose 6-phosphate or dihydroxyacetone phosphate, indicating that this protein might be the amyloplastic hexosephosphate translocator.

Effect of atractylosides, palmitoyl coenzyme A, and anion transport inhibitors on translocation of nucleotide sugars and nucleotide sulfate into Golgi vesicles.

The effect of palmitoyl coenzyme A, atractylosides, and anion transport inhibitors on translocation into rat liver Golgi vesicles of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), CMP-N-acetylneuraminic acid, and GDP-fucose was studied. Translocation of the above three nucleotide derivatives was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50% inhibition required 10-20 microM DIDS). The inhibition of translocation of PAPS by DIDS was used to demonstrate that sulfation of macromolecules within Golgi vesicles is preceded by translocation of PAPS into the vesicles. Palmitoyl coenzyme A, at concentrations below its critical micellar concentration, specifically inhibited translocation into Golgi vesicles of PAPS but not CMP-NeuAc and GDP-fucose. Inhibition of PAPS translocation by 50% required 9 microM palmitoyl coenzyme A. Translocation of PAPS but not of CMP-NeuAc or GDP-fucose was also inhibited by atractyloside or carboxyatractyloside with 50% inhibition requiring 15 microM either glycoside. This pattern of inhibition suggests structural similarities between the putative translocator of PAPS in Golgi membranes and the ATP/ADP translocator of mitochondria.

Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts.

The relationship between the electrochemical proton gradient (delta mu-H+) and the electrochemical gradient for biogenic amines (delta mu-A) was investigated in isolated chromaffin ghosts free of endogenous components and gradients. The addition of ATP to a ghost suspension resulted in the generation of a large proton concentration gradient (delta pH), acidic inside (measured by [14C]-methylamine distribution), and a large proton electrical gradient (delta psi), positive inside (measured by [14C]-thiocyanate distribution). In the presence of this large electrochemical proton gradient, the accumulation of [14C]5-hydroxytryptamine (serotonin) and other biogenic amines rapidly reached an apparent steady state level. Collapse of the proton gradients after steady state levels were achieved resulted in the efflux of the accumulated amines. Uptake in the presence of a delta psi alone produced an amine gradient equal to the magnitude of the delta psi, while in the presence of a delta pH alone biogenic amine distribution was equal to twice the magnitude of the delta pH. Using additions of ammonia or thiocyanate, it was possible to vary the magnitude of the electrochemical proton gradient over a wide range of values; the driving force for amine accumulation under these conditions was found to be equal to delta psi--2Z delta pH (where Z = 2.3 RT/F). The results, which provide unequivocal evidence for the primary role of the electrochemical proton gradient in the active transport of biogenic amines, are consistent with a model based on the chemiosmotic hypothesis, of an obligatory coupling of amine influx and proton efflux, mediated via a putative reserpine-sensitive translocator. Measurement of the stoichiometry of this coupling of H+ and amines permits conclusions concerning the molecular mechanism of amino accumulation.