ABSTRACT
Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.
Subject(s)
Evolution, Molecular , Mitochondrial Proteins/chemistry , Peptide Hydrolases/chemistry , Protozoan Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Animals , Arabidopsis/classification , Arabidopsis/enzymology , Arabidopsis/genetics , Conserved Sequence , Euglena gracilis/classification , Euglena gracilis/enzymology , Euglena gracilis/genetics , Euglena longa/classification , Euglena longa/enzymology , Euglena longa/genetics , Gene Expression , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Leishmania major/classification , Leishmania major/enzymology , Leishmania major/genetics , Mice , Mitochondria/enzymology , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Phylogeny , Protein Domains , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Trypanosoma brucei brucei/classification , Trypanosoma brucei brucei/geneticsABSTRACT
The measurement of respiratory chain enzyme activities is an integral part of basic research as well as for specialized examinations in clinical biochemistry. Most of the enzymes use ubiquinone as one of their substrates. For current in vitro measurements, several hydrophilic analogues of native ubiquinone are used depending on the enzyme and the workplace. We tested five readily available commercial analogues and we showed that Coenzyme Q2 is the most suitable for the measurement of all tested enzyme activities. Use of a single substrate in all laboratories for several respiratory chain enzymes will improve our ability to compare data, in addition to simplifying the stock of chemicals required for this type of research.
