Alternative oxidase activity is necessary for growth in high osmolarity media.

<p>(A) Cells were grown in YPD media with and without NaCl, KCl or sorbitol (white bars). Some plates were supplemented with 3 mM SHAM (gray bars) to determine the effect of Aox on hyperosmotic media. Samples were spread on YPD plates at 28°C and colonies were counted after 2 days. (B) Representative plates of serially diluted samples to show the effect of 3 mM SHAM on cell grown in the presence of the indicated osmolites. Ctrl, YPD media, Na⁺, YPD plus 0.6 M NaCl, S, YPD, YPD media plus 1.2 M sorbitol, K⁺, YPD media plus 0.6 M KCl. (C-D) Compatible solute production in the cells grown in the presence of the indicated osmolites. Cells grown in the indicated media were lysed and extracts assayed for trehalose (C) and glycerol (D) content. * <i>p</i> < 0.05 respect to control cells.</p

High osmolarity promotes higher resistance to oxidative stress.

<p>(A) Hydrogen peroxide production rates in isolated mitochondria. Isolated mitochondria (0.5 mg/mL) from cells in each experimental condition were assayed for hydrogen peroxide production using 1 mM NADH as a respiratory substrate. White bars, basal hydrogen peroxide production. Light gray bars, hydrogen peroxide production in the presence of 100 μM SHAM. Dark gray, hydrogen peroxide production in the presence of 1 μM antimycin A. (B) Cells grown YPD media (white bars) or YPD+SHAM (3 mM, gray bars) were treated with 10 mM hydrogen peroxide for 10 minutes, samples were plated on YPD and incubated at 29°C for 2 days. Colonies were manually counted. *<i>p</i> < 0.05 respect to basal oxidant production (white bars, panel A), #<i>p</i> < 0.05 respect to oxidant production in the presence of SHAM (Light gray bars, panel A) For panel B, *<i>p</i> < 0.05 respect to control cells with SHAM. # <i>p</i> < 0.05 respect to control without SHAM.</p

Simplified representation of the respiratory chain of <i>Debaryomyces hansenii</i>.

<p><i>Debaryomyces hansenii</i> has a proton-pumping complex I, a canonical proton-pumping cytochrome pathway (complex III-IV) and a cyanide-resistant terminal oxidase (Aox). For simplicity, the alternative NADH dehydrogenase and glycerol-3-phosphate dehydrogenase described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169621#pone.0169621.ref009" target="_blank">9</a>] are not depicted. Electrons flow from complex I to ubiquinone and at this point the respiratory chain is branched and electrons flow to complex III and the Aox. Thus, two electron pathways are able to sustain a protonmotive force: Complex I-Aox and Complex I-III-IV.</p

Increases in osmolarity in glucose media promote cyanide-resistance respiration.

<p>(A) Cyanide-resistant respiration in cells grown in YPD. Cells (1 mg.mL^-1) were incubated in respiration buffer. To induced Aox-dependent respiration, 25 mM glucose and 0.5 mM potassium cyanide (KCN) were included in the incubation media. (B-D) Densitometry analysis of the expression of the Aox (Panel B) and cytochrome <i>c</i> oxidase subunit III (CoxIII, Panel C) in glucose media in the presence of 0.6 M NaCl, 1.2 M sorbitol or 0.6 M KCl. (D) Representative blots of Aox and CoxIII expression.</p

Aox sustains a membrane potential (ΔΨ) when coupled to complex I.

<p>(A) Representative plot of mitochondrial membrane potential measurements in intact cells grown in glucose media Where indicated, 25 mM glucose, 0.5 μM antimycin A (AA), 3 mM SHAM and 10 μM CCCP (U) were added. Cells were incubated in regular respiration media at a final concentration of 1 mg.mL^-1 (B) Aox-dependent ΔΨ measured as the difference between maximum fluorescence (glucose) and the fluorescence after Aox inhibition (SHAM). (C-E) Representative ΔΨ plots of isolated mitochondria from cells grown in YPD. (C) Complex I-dependent ΔΨ (10 mM pyruvate/malate/citrate each). Traces: Black, control without inhibitors. Red, Aox-sustained ΔΨ, in the presence of 100 μM cyanide (addition where indicated). Green/Blue, contribution of the cytochromes to the ΔΨ after the addition of 100μM salicylhydroxamic acid (SHAM, green) and 1 μM propyl gallate (PG, blue). (D) ΔΨ- dependent on Aox. Traces: Black, control without inhibitors. Blue/Red after addition of 100μM SHAM (blue) and 1 mM PG (Green) respectively. (F) Succinate/rotenone (10 mM/ 1 μM) dependent ΔΨ. Traces: Blue, complete collapse of ΔΨ after 100 μM cyanide addition. Black/Red, inhibition of Aox by SHAM (black) or PG (red) did not exert any effect on the ΔΨ, complete collapse of ΔΨ under these conditions was obtained after addition of 100 μM cyanide. At the end of all plots, 1 μM CCCP (U) was added to completely collapse the ΔΨ. <i>* p</i> < 0.05 respect to control cells.</p

Sodium is incorporated into cells without loss of cell volume.

<p>(A-B) Cells grown in YPD media until logarithmical phase were washed and suspended in buffer (20 mM Hepes pH 6.8) (A) or buffer supplemented with 0.6 M NaCl (B) and cells analyzed by flow cytometry. Letters A and B inside these figures represent the two subpopulations observed by flow cytometry. (C) Percentage of cells in each subpopulation for each treatment. (D Cellular sodium uptake under the same conditions used in the flow cytometry assays. Representatives traces. Black trace, control (cells in 20 mM Hepes pH 6.8). Red, cells in buffer with subsequent addition of 0.6 M NaCl. Blue trace, as in black trace but where indicated 10μM gramicidin plus 10 μM alamethicin was added to the incubation media. Glucose (25 mM) was included in all the experiments to energize the cells. * p < 0.05 respect to control (YPD) samples.</p