Abstract
Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in both the oxidative and nitrosative stress responses in Saccharomyces cerevisiae. Previous studies have shown that the expression of YHB1 is optimal under normoxic or hyperoxic conditions, yet respiring yeast cells have low levels of reduced YHb pigment as detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells. Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships between respiration, YHb level, and intracellular location. We have found that although intact respiration-proficient cells lack a YHb CO spectral signature, cell extracts from these cells have both a YHb CO spectral signature and nitric oxide (NO) consuming activity. This suggests either that YHb cannot be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells. By using an anti-YHb antibody and CO difference spectroscopy and by measuring NO consumption, we have found that YHb localizes to two distinct intracellular compartments in respiring cells, the mitochondrial matrix and the cytosol. Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome. The findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function.
Highlights
Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in both the oxidative and nitrosative stress responses in Saccharomyces cerevisiae
Despite the absence of a carbon monoxide (CO) spectral signature assignable to YHb in JM43 cells, we have found that YHB1 mRNA levels in JM43 and JM430 are essentially identical (Fig. 1B)
This study presents three novel findings concerning the YHb flavohemoglobin in yeast
Summary
Media, and Growth Conditions—The following strains of S. cerevisiae were used: JM43 (MAT␣ his580 trp289 leu 112 ura3-52 [rhoϩ]) [20]; JM430, an isochromosomal respiration-deficient derivative of JM43 that lacks a functional mitochondrial genome [16]; JM43GD5ab (JM43 with cox5b::LEU2 cox5aD::URA3), an isogenic derivative of JM43 carrying gene disruptions in the COX5a and COX5b genes [21]; and DR11 (JM43 with yhb1::URA1), an isogenic derivative of JM43 carrying a YHB1 gene disruption [19]. The supernatant was adjusted to 65% ammonium sulfate, and the suspension was incubated at 4 °C for 15 min with stirring and centrifuged at 27,000 ϫ gmax for 15 min at 4 °C. NO consumption of whole cell extracts or mitochondrial or cytosolic fractions was measured in 2 ml of phosphate-buffered saline (80 mM Na2HPO4, 20 mM NaH2PO4, and 100 mM NaCl), 250 M NADH, and 0.1 mM EDTA after the addition of 2 M NO from an NO-saturated solution of distilled water. Miscellaneous Methods—Protein determination was performed using either the Lowry assay [31] or the BCA assay (Pierce Biotechnology) with bovine serum albumin as a standard
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