Abstract
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.
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Acknowledgements
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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Supplementary Fig. 1
Mitochondrial intactness assays. a) Mitochondrial oxygen consumption in the presence of succinate by exogenous cytochrome c in different strains. The OCR (state 3) following the addition of exogenous cytochrome c is presented for 7 biological replicates of all strains except ΔN2-12Por, for which there were 8. Average values are presented and error bars indicate standard deviation; there were no statistically significant differences among the preparations, as assessed using the students’ t-test. Increases of less than 20% indicate high quality mammalian mitochondria suitable for bioenergetics assays (Banh et al. 2015). b) Protease treatment of mitochondria. Two samples of mitochondria were mixed in SR buffer at 30 °C in the Oroboros respirometry chamber. In one chamber the standard cytochrome c-based intactness assay was carried out. For the experiments shown, the ratios of OCR ± cytochrome c addition were WT: 1.2; LoPo: 1.1; ΔPor-1: 1.2; Δ2-12Porin: 1.1. From the other chamber, two 500 μl samples were taken at each time point; one was kept on ice (-protK) and the other was protease-treated (+ protK) on ice as described in Materials and Methods. Lanes 1, samples taken prior to the beginning of the experiment (unmixed); lanes 2, samples taken at the same time as the addition of cytochrome c (after 10–15 min of mixing) and lanes 3, samples taken at the end of the experiment (15–20 min). Proteins were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies against Tom70 (70-kDa subunit of the translocase of the outer membrane), CCHL (cytochrome c heme lyase; 38 kDa) and MPP (α-subunit of the mitochondrial-processing peptidase, 63 kDa). (PPTX 3.10 mb)
Supplementary Figure 2
OCR in the presence and absence of TMRM. Oxygen consumption rates were measured for mitochondria from each strain, following the sequential addition of GMS, ADP, and oligomycin. Data obtained in the presence (grey bars) and absence (white bars) of TMRM are presented as average OCR for 3 biological replicates of ΔN2-12Por and LoPo and 4 biological replicates for WT and ΔPor-1; error bars indicate standard deviations. The star indicates a statistically significant difference (p > 0.05) between the OCR in the presence and absence of TMRM (Student’s t test). (PPTX 151 kb)
Supplementary Figure 3
Membrane potential and oxygen consumption rates of mitochondria respiring on complex I (GM) and complex II (S) and all three (GMS) substrates. OCR (a) and Δψ (b) data were collected simultaneously by combined respirometry and fluorescence in the presence of the indicated substrates. a) Oxygen consumption rates in mitochondria. OCR was measured as described in materials and methods, following sequential addition of substrate (state 2), ADP (state 3) and oligomycin (state 4o). b) Mitochondrial ΔΨ as estimated by quenching of the fluorescent dye TMRM. For each of the indicated strains, state 2 data indicate the relative TMRM fluorescence in the presence of substrate (ΔFA/FT × 100; see Fig. 3), state 3 data show the degree of quenching of fluorescence (depolarization of mitochondria) in the presence of ADP (ΔFB/FT × 100), and state 4o data represent the quenching observed in the presence of oligomycin (ΔFC/FT × 100). The data presented are ranges obtained from 2 biological replicates, each of which was examined under all three substrate combinations. The top of the bar indicates the lowest value obtained, and the top of the error bar indicates the highest value. Given the small sample size, statistical support for apparent differences cannot be obtained. Black, WT; white, LoPo; grey, ΔPor-1; hatched, ΔN2-12Por. (PPTX 6.91 mb)
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Shuvo, S.R., Wiens, L.M., Subramaniam, S. et al. Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria. J Bioenerg Biomembr 51, 341–354 (2019). https://doi.org/10.1007/s10863-019-09807-6
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DOI: https://doi.org/10.1007/s10863-019-09807-6