Monothiol glutaredoxin-1 is an essential iron-sulfur protein in the mitochondrion of African trypanosomes

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx). 1-C-Grx1 occurs exclusively in the mitochondrion, and 1-C-Grx2 and -3 are predicted to be mitochondrial and cytosolic proteins, respectively. All three 1-C-Grx are expressed in both the mammalian bloodstream and the insect procyclic form of Trypanosoma brucei, with the highest levels found in stationary phase and starving parasites. In the rudimentary mitochondrion of bloodstream cells, 1-C-Grx1 reaches concentrations above 200 microm/subunit. Recombinant T. brucei 1-C-Grx1 exists as a noncovalent homodimer, whereas 1-C-Grx2 and 1-C-Grx3 are monomeric proteins. In vitro, dimeric 1-C-Grx1 coordinated an H(2)O(2)-sensitive [2Fe-2S] cluster that required GSH as an additional ligand. Both bloodstream and procyclic trypanosomes were refractory to down-regulation of 1-C-Grx1 expression by RNA interference. In procyclic parasites, the 1-c-grx1 alleles could only be deleted if an ectopic copy of the gene was expressed. A 5-10-fold overexpression of 1-C-Grx1 in both parasite forms did not yield a growth phenotype under optimal culture conditions. However, exposure of these cells to the iron chelator deferoxamine or H(2)O(2), but not to iron or menadione, impaired cell growth. Treatment of wild-type bloodstream parasites with deferoxamine and H(2)O(2) caused a 2-fold down- and up-regulation of 1-C-Grx1, respectively. The results point to an essential role of the mitochondrial 1-C-Grx1 in the iron metabolism of these parasites.     

Crystal structure of the Trypanosoma cruzi trypanothione reductase·mepacrine complex

The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 Å resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser-109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp-21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent-mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease. © 1996 Wiley-Liss, Inc.     

Photoaffinity cross-linking of oligomycin-sensitive ATPase from beef heart mitochondria by 3′-arylazido-8-azido ATP

Photoaffinity cross-linking of the oligomycin-sensitive ATPase from beef heart mitochondria by 3′-arylazido-8-azido ATP results in a nucleotide specific formation of a cross-link between α and/or β subunits. Moreover a nucleotide independent decrease of the heterogeneous 29–31 kd protein band is observed. This decrease can be reduced by addition of 2,4-dinitrophenol or 2-azido-4-nitrophenol.     

Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.     

The Extracellular δ-Domain is Essential for the Formation of CD81 Tetraspanin Webs

CD81 is a ubiquitously expressed member of the tetraspanin family. It forms large molecular platforms, so-called tetraspanin webs that play physiological roles in a variety of cellular functions and are involved in viral and parasite infections. We have investigated which part of the CD81 molecule is required for the formation of domains in the cell membranes of T-cells and hepatocytes. Surprisingly, we find that large CD81 platforms assemble via the short extracellular δ-domain, independent from a strong primary partner binding and from weak interactions mediated by palmitoylation. The δ-domain is also essential for the platforms to function during viral entry. We propose that, instead of stable binary interactions, CD81 interactions via the small δ-domain, possibly involving a dimerization step, play the key role in organizing CD81 into large tetraspanin webs and controlling its function.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

Uptake of thyroid hormone by isolated rat liver cells

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent K_t value of 52±22 nM and a system with low affinity with an apparent K_t value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.     

Osmotische Resistenz und Turgordehnung der Zoosporen einiger mariner Cladophoren

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