Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1.
African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.
Filser
Michael
M
Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 504, D-69120 Heidelberg, Germany.
Comini
Marcelo A
MA
Molina-Navarro
Maria M
MM
Dirdjaja
Natalie
N
Herrero
Enrique
E
Krauth-Siegel
R Luise
RL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D000431:Ethanol / Q000031:analogs & derivatives / Q000378:metabolism; D005816:Genetic Complementation Test; D054477:Glutaredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D019163:Reducing Agents / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D003062:Codon; D016054:DNA, Protozoan; D004220:Disulfides; D054477:Glutaredoxins; D007328:Insulin; D011994:Recombinant Proteins; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D000431:Ethanol; C031319:2-hydroxyethyl disulfide; D010088:Oxidoreductases