Abstract
Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ620469, AJ620470, and AJ620471
To examine changes in mitochondrial biochemistry of Euglena during the shift from aerobic to anaerobic conditions, we investigated changes in ubiquinone and rhodoquinone content and isolated mitochondria from cells grown under both conditions to analyze their protein content via twodimensional electrophoresis
Activities are expressed in nmol1⁄7mgϪ11⁄7minϪ1 for crude extract and mitochondrial fraction of E. gracilis cells grown under aerobic and anaerobic conditions (ϮS.D.)
Summary
Medium and Culture Conditions—E. gracilis strain Z (SAG 1224 – 5/25 collection of algae Gottingen) for isolation of mitochondria and subsequent analysis by two-dimensional PAGE was cultured as described previously [6]. Calibration of the liquid chromatography mass spectrometry method was performed using UQ9 standards (Sigma) and RQ9 standards (isolated from A. suum according to Bligh and Dyer [33] and purified as described by Van Hellemond et al [34]), which resulted in linear response curves between 0.1 and 100 pmol for RQ9 and between 0.35 and 350 pmol for UQ9. Identification of PDH Subunits E1␣, E2, and E3—Standard molecular methods, nucleic acid isolation, cDNA synthesis, and cloning in ZAPII were performed as described previously [36, 37]. Phylogenetic inference was performed using NeighborNet planar graphs [40] of protein LogDet distances [41]; graphs were displayed with SplitsTree package, version 3.2 [42]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
