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CARYN E. OUTTEN, PhD
Outten
CE, Falk RL, Culotta VC. Cellular factors required for
protection from hyperoxia toxicity in Saccharomyces
cerevisiae. Biochem J. 2005 May 15;388(Pt 1):93-101.
Prolonged exposure to hyperoxia
represents a serious danger to cells, yet little is known
about the specific cellular factors that affect hyperoxia
stress. By screening the yeast deletion library, we have
identified genes that protect against high-O2 damage. Out of
approx. 4800 mutants, 84 were identified as hyperoxia-sensitive,
representing genes with diverse cellular functions,
including transcription and translation, vacuole function,
NADPH production, and superoxide detoxification. Superoxide
plays a significant role, since the majority of hyperoxia-sensitive
mutants displayed cross-sensitivity to superoxide-generating
agents, and mutants with compromised SOD (superoxide
dismutase) activity were particularly vulnerable to
hyperoxia. By comparison, factors known to guard against
H2O2 toxicity were poorly represented amongst hyperoxia-sensitive
mutants. Although many cellular components are potential
targets, our studies indicate that mitochondrial glutathione
is particularly vulnerable to hyperoxia damage. During
hyperoxia stress, mitochondrial glutathione is more
susceptible to oxidation than cytosolic glutathione.
Furthermore, two factors that help maintain mitochondrial
GSH in the reduced form, namely the NADH kinase Pos5p and
the mitochondrial glutathione reductase (Glr1p), are
critical for hyperoxia resistance, whereas their cytosolic
counterparts are not. Our findings are consistent with a
model in which hyperoxia toxicity is manifested by
superoxide-related damage and changes in the mitochondrial
redox state.
Outten CE, Culotta VC. Alternative
start sites in the Saccharomyces cerevisiae GLR1 gene are
responsible for mitochondrial and cytosolic isoforms of
glutathione reductase. J Biol Chem. 2004 Feb
27;279(9):7785-91.
To combat oxidative damage, eukaryotic
cells have evolved with numerous anti-oxidant factors that
are often distributed between cytosolic and mitochondrial
pools. Glutathione reductase, which regenerates the reduced
form of glutathione, represents one such anti-oxidant
factor, yet nothing is known regarding the partitioning of
this enzyme within the cell. Using the bakers' yeast
Saccharomyces cerevisiae as a model, we provide evidence
that a single gene, namely GLR1, encodes both the
mitochondrial and cytosolic forms of glutathione reductase.
A deletion in GLR1 drastically increases levels of oxidized
glutathione in these two subcellular compartments. The GLR1
gene has two inframe start codons that are both used as
translation initiation sites. Translation from the first
codon generates the mitochondrial form that includes a
mitochondrial targeting signal, whereas translation from the
second codon produces the cytosolic form that lacks this
sequence. Our results indicate that the sequence context of
the two AUG codons influences the efficiency of translation
initiation at each site, which in turn affects the relative
levels of cytosolic and mitochondrial Glr1p. This method of
subcellular distribution of glutathione reductase may be
conserved in mammalian cells as well.
Changela A, Chen K, Xue Y, Holschen J,
Outten CE, O'Halloran TV, Mondragon A. Molecular basis of
metal-ion selectivity and zeptomolar sensitivity by CueR.
Science. 2003 Sep 5;301(5638):1383-7.
The earliest of a series of copper efflux
genes in Escherichia coli are controlled by CueR, a member
of the MerR family of transcriptional activators.
Thermodynamic calibration of CueR reveals a zeptomolar
(10(-21) molar) sensitivity to free Cu+, which is far less
than one atom per cell. Atomic details of this extraordinary
sensitivity and selectivity for +1transition-metal ions are
revealed by comparing the crystal structures of CueR and a
Zn2+-sensing homolog, ZntR. An unusual buried metal-receptor
site in CueR restricts the metal to a linear, two-coordinate
geometry and uses helix-dipole and hydrogen-bonding
interactions to enhance metal binding. This binding mode is
rare among metalloproteins but well suited for an
ultrasensitive genetic switch.
Outten CE, Culotta VC. A novel NADH
kinase is the mitochondrial source of NADPH in Saccharomyces
cerevisiae. EMBO J. 2003 May 1;22(9):2015-24.
Mitochondria require NADPH for
anti-oxidant protection and for specific biosynthetic
pathways. However, the sources of mitochondrial NADPH and
the mechanisms of maintaining mitochondrial redox balance
are not well understood. We show here that in Saccharomyces
cerevisiae, mitochondrial NADPH is largely provided by the
product of the POS5 gene. We identified POS5 in a
S.cerevisiae genetic screen for hyperoxia-sensitive mutants,
or cells that cannot survive in 100% oxygen. POS5 encodes a
protein that is homologous to NAD(+) and NADH kinases, and
we show here that recombinant Pos5p has NADH kinase
activity. Pos5p is localized to the mitochondrial matrix of
yeast and appears to be important for several NADPH-requiring
processes in the mitochondria, including resistance to a
broad range of oxidative stress conditions, arginine
biosynthesis and mitochondrial iron homeostasis. Pos5p
represents the first member of the NAD(H) kinase family that
has been identified as an important anti-oxidant factor and
key source of the cellular reductant NADPH.
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