Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: interactions of thioredoxin with elongation factor 1alpha and yeast alcohol dehydrogenase.
Thioredoxins (Trx) are ubiquitous dicysteine proteins capable of modulating enzymes and other cellular targets through specific disulfide-dithiol redox changes. They are unique in that a large number of very diverse metabolic systems are addressed and redox-regulated in bacteria, animal, and plant cells, but the finite number of thioredoxin interaction partners is still unknown. Two-hybrid methodology should provide a rational way to establish thioredoxin functions in a given organism. We report a search for physiological target proteins of thioredoxin1 in the social amoeba Dictyostelium discoideum , which possesses three developmentally regulated thioredoxin genes, all of which lack functional characterisation. A two-hybrid approach identified at least seven bona fide thioredoxin partners, including oxidoreductases, proteins of the ribosomal translation apparatus, and the cytoskeletal protein filopodin. With the exception of ribonucleotide reductase, none of these systems had previously been linked to specific redox modulation. Molecular interactions in two of the new thioredoxin/target protein couples were verified by biochemical studies: (1) thioredoxin1 and the abundant elongation factor 1alpha from D. discoideum form the mixed heterodisulfide characteristic of the thioredoxin mechanism of action; and (2) reduced thioredoxin, but not glutathione, strongly inhibits yeast alcohol dehydrogenase catalysis of ethanol oxidation.
Brodegger
Thomas
T
Department of Biochemistry, Natural Sciences Faculty, University of Kassel, D-34109 Kassel, Germany.
Stockmann
Anja
A
Oberstrass
Jürgen
J
Nellen
Wolfgang
W
Follmann
Hartmut
H
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015723:Gene Library; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013879:Thioredoxins / Q000235:genetics / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D004791:Enzyme Inhibitors; D020648:Peptide Elongation Factor 1; D013879:Thioredoxins; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; D012264:Ribonucleotide Reductases