Scheme of the plasma membrane redox system where electrons move from internal electron donor such as NADH to external oxidants through the reduction of Q to QH₂.

(A) NQR1 shows an early expression during low glucose culture conditions. Wild type cells (BY4741) were inoculated in YPD media containing several glucose concentrations (0.5, 2 and 10%). At the indicated points samples were taken to measure NQR1 expression by RT-PCR. Data corresponds to the average of two independent experiments and each point was calculated with at least three reactions. Data are normalized using ACT1 expression. Control point (0 h) corresponds with the cells used to inoculate the different cultures that were grown in YPD 10% 16 h (B) Plasma membrane samples purified from BY4741 cells cultured in YPD at several glucose concentrations (0.5, 2 and 10%) were analyzed by western blot using a polyclonal anti-NQR1p antibody. The analysis with anti-Gas1p polyclonal antibody was performed as loading control. Cultures were inoculated at 0.1 U OD 660nm/ml and cells were harvested at 10 and 24 hours to purify the plasma membrane. (C) Densitometric analysis of Nqr1p expression normalized with the Gas1p expression. Arbitrary units were calculated for each sample and the highest level was used as 100% in each blot. (D) The same plasma membrane samples were subjected to the measure of the activity of plasma membrane redox system NADH-Q reductase. Activity is expressed as the average ± SD of at least three measurements. ^a Data are significantly different compared with other data (p< 0.001). ^b Data are significantly different compared with YPD 10% data (p< 0.001) (E) The same plasma membrane samples were subjected to HPLC-ECD to measure the levels of Q₆. The HPLC peaks corresponding to the reduced Q₆ (Q₆H₂) were integrated and compared with total Q₆ in order to calculate the proportion against total Q₆. ^a Data are significantly different compared with other data (p< 0.001).

(A) Fluorescence microscopy indicates a double localization in plasma membrane and karmellae. Wild type cells transformed with the pNQR1 or pYES2 vector were cultured in galactose (0.05% or 2%) and glucose 2%. pNQR1 is the pYES2 empty vector harboring the coding sequence of the YML125c gene (NQR1) fused with V5 epitope controlled by the GAL promoter. Cells were subjected to epifluorescence microscopy analysis using V5 antibody as primary and anti-mouse labeled with FITC as secondary. Cell nuclei were labeled with DAPI. All images were obtained with the same magnification (×1000). (B) Wild type cells BY4741 over expressing NQR1 analyzed with confocal microscopy. Is possible appreciate with more detail the karmellae structure and the plasma membrane. (C) Mitochondrial localization of NQR1p after over expression is an artifact of mitochondrial purification process. The nuclear copy of the NQR1 gene was labeled with the c-Myc epitope after the insertion of a kanamycin cassette fused in frame. Cell subfractionation performed with labeled cells was analyzed by western blotting using as primary antibodies Gas1p (plasma membrane and DIGs), c-Myc, Pma1p (plasma membrane), Porin (mitochondrial outer membrane) and Kar2p (ER). Western blots corresponds to TF (total fraction), PM (plasma membrane), DIGs (detergent insensitive glycolipid-enriched fractions), Mito (mitochondria), P30 (pellet from 30,000 rpm centrifugation), P100 (pellet from 100,000 rpm centrifugation) and S100 (supernatant from 100,000 rpm centrifugation). (D) Plasma membranes samples from wild type yeast strains strain harboring the control vector (pYES2) and the same vector containing the gene NQR1 (pNQR1) and cultured in several amounts of galactose (0.05% or 2%) were used to measure three typical activities of the plasma membrane redox system. Results are expressed as the average of at least three assays ± SD. ^a Data are significantly different compared with other data (p< 0.001), ^b Data are significantly different compared only with pYES2 2% data (p< 0.001).

(A) Plasma membrane samples from control (pYES2) and NQR1 over expressing cells (pNQR1) were subjected to lipid extraction and separation by HPLC to analyze the levels of Q₆ in both redox states. Some samples were previously incubated in presence of 0.2 mM NADH. (B) The areas of HPLC peaks corresponding to the reduced Q₆ (Q₆H₂) were integrated and compared with total Q₆ in order to calculate the proportion against total Q₆. PM: Plasmas membrane sample. Data correspond to the integration of three chromatograms from the same plasma membrane sample. Data from plasma membrane obtained from cells expressing NQR1 (pNQR1) were significantly higher (p<0.001) that control samples. Data from plasma membrane samples incubated with NADH were significantly higher (p<0.001) that non incubated samples. (C) The wild type yeast strain BY4741 harboring the control vector (pYES2) and the same vector containing the gene NQR1 (pNQR1) were cultured in 2% galactose. At the indicated times were collected samples to quantify separately the level of pyridine nucleotides. Results correspond to the pool of pyridine nucleotides and are expressed as the average of three assays ± SD and are representative of a set of two experiments. Inside the bars is indicated the ratio NAD⁺/NADH. ^a The pool of pyridine nucleotides at 10 and 24 hours was significantly higher in pYES2 cells compared to pNQR1 cells (p<0.001).

(A) Chronological life span assay. Liquid cultures containing galactose as carbon source were inoculated with the same number of cells. After 5 days of growth, samples of each culture were taken to measure the cell viability in YPD plates. The first day was used as control (100%). Average life was BY4741:pYES2 (6.38), BY4741:pNQR1 (11.13), BY4741 SIR2∷KMX4:pYES2 (8.11), BY4741 SIR2∷KMX4:pNQR1 (11.55). The survival log rank analysis shown a p<0.001 with the exception of BY4741:pNQR1/SIR2∷KMX4:pNQR1 that was p=0.244. The plots show a representative experiment repeated 3 times with similar results. (B) Replicative life span analysis. Average life was BY4741:pYES2 (28.07), BY4741:pNQR1 (36.75), BY4741 SIR2∷KMX4:pYES2 (16.52), BY4741 SIR2∷KMX4:pNQR1 (20.27) The survival log rank analysis shown a p<0.001 with the exception of BY4741 SIR2∷KMX4:pYES2/BY4741 SIR2∷KMX4:pNQR1 that was p=0.004. The analysis was carried out twice independently with more than 45 cells for each strain each time. Representative results are shown.

(A) Oxygen consumption was measured in wild type cells (BY4741 strain) harboring the control vector (pYES2) or pNQR1 in a continuous method in parallel. (B) Same cells as previous experiment were used to measure the ethanol production during the growth. Was measured the ethanol concentration in the culture media. (C) Mitochondrial activities measured in wild type cells (BY4741) harboring the control vector (pYES2) or pNQR1. Results show average values and SD for at least three assays and were expressed as nmol/mg mitochondrial protein.min. Data from mitochondria obtained from cells expressing NQR1 (pNQR1) were significantly higher (p<0.001) that control samples. CS, citrate synthase; I, Complex I, NADH-DCPIP reductase; II, Complex II Succinate-DCPIP reductase; III, bc₁ complex, decyl ubiquinone-cytochrome c reductase; IV, complex IV cytochrome c oxidase and I+III, NADH-cytochrome c reductase.

(A) Several strains harboring the control plasmid pYES2 or the pNQR1 plasmid that were spotted in 10-fold dilutions in SDc - ura plates with 2% glucose or 2% galactose in order to analyze the requirement of Q₆ to produces life span extension. BY4741, wild type; ΔCOQ2, defective in the Q₆ synthesis; ΔCOR1, defective in the bc₁ complex; ΔATP2, defective in the ATP synthase complex. (B) The previous experiment was carried out in liquid media in order to complement the respiration deficiency with 2 μM of exogenous Q₆. The symbol (+) indicates the rate of growth.

A model to approach the role of NQR1 in NADH oxidation to promote NAD⁺ raise. This is connected to NAD⁺ reduction of ADH2 to decrease ethanol accumulation.