Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria
The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria.
KeywordsVDAC Mitochondrial porin Neurospora crassa Alternative oxidase Membrane potential Reactive oxygen species
This work was supported by Discovery Grants (DG) from the Natural Sciences and Engineering Research Council of Canada (NSERC) to DAC and JRT, the Canada Research Chairs (CRC) program to JRT and the Faculty of Science (DC). SRS acknowledges support from the Faculty of Graduate Studies at the University of Manitoba and DG funds awarded to DAC. LMW was supported by DG and CRC funds to JRT. The authors thank Mr. Erwin J. Taguiam, Department of Microbiology, for excellent technical assistance, Dr. Frank Nargang, University of Alberta, for antibodies, and Dr. Richard Sparling, Department of Microbiology, for very valuable discussions and reading of the manuscript.
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