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Role of IscS in Fe‐S cluster assembly in Trypanosoma brucei
Author(s) -
SMÍD O.,
VONDRUSKOVÁ E.,
VILÍMOVÁ V.,
SUT′ÁK R.,
LUKE J.,
TACHEZY J.
Publication year - 2005
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2005.05202003_5_7.x
Subject(s) - aconitase , trypanosoma brucei , biology , cytosol , mitochondrion , biochemistry , iron–sulfur cluster , rna interference , microbiology and biotechnology , enzyme , rna , gene
Despite the significance of proteins containing iron‐sulfur cluster (Fe–S proteins), the processes of Fe–S cluster assembly and maturation of Fe–S proteins are poorly understood. However, several key proteins involved in the assembly have been identified, notably IscS, a cystein desulfurase, which provides sulfur for Fe–S cluster and IscU, a metallochaperone acting as a scaffold for cluster assembly. In this work, we studied the process of Fe–S cluster biosynthesis in Trypanosoma brucei by identifying the homologue of IscS in the T. brucei (TbIscS). To address the function of TbIscS, we inhibited its expression by means of RNA interference (RNAi). After RNAi induction, generation time of the TbIscS knock‐down cell line was significantly prolonged. All types of mitochondrial ATP production in the cells were severely affected. Analysis of glucose metabolism end products determined pyruvate as major excreted metabolite of the induced cells, while the uninduced cells produced only small amount of this glycolytic end product. These data demonstrate that mitochondrial metabolism is impaired in cells with TbIscS knocked down. To test whether the observed phenomena were results of Fe–S cluster assembly disruption, we examined the Fe–S cluster‐dependent activity of aconitase. This enzyme is localized in its active form in mitochondrion as well as in cytosol of T. brucei . After RNAi induction we observed the reduction of aconitase activity in both compartments (approx. 70% reduction in cytosol, approx. 30% in mitochondria). Western blots together with the EPR analysis showed that the reduction in cytosolic activity was due to impaired Fe–S cluster formation, while decrease in aconitase activity in mitochondria corresponded to the reduced level of the protein.