Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata.
Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.
Breek
C K
CK
Dept. of Biochemistry, University of Amsterdam, The Netherlands.
Speijer
D
D
Dekker
H
H
Muijsers
A O
AO
Benne
R
R
D000818:Animals; D015153:Blotting, Western; D016831:Crithidia fasciculata / Q000201:enzymology* / Q000648:ultrastructure; D051546:Cyclooxygenase 2; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology; D008928:Mitochondria / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry / Q000276:immunology; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000276:immunology; D016053:RNA, Protozoan / Q000096:biosynthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D016053:RNA, Protozoan; D010544:Peroxidases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D003576:Electron Transport Complex IV; D003545:Cysteine