Yeast Flavohemoglobin , a Nitric Oxide Oxidoreductase, is ... · heme binding domain, the “myoglobin fold” (2). Aside from these common features hemoglobins Aside from these common

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Yeast Flavohemoglobin, a Nitric Oxide Oxidoreductase, is Located both in the Cytosol and Mitochondrial Matrix

Effects of Respiration, Anoxia, and the Mitochondrial Genome on Its Intracellular Level and Distribution

Nina Cassanova, Kristin M. O’Brien‡, Brett T. Stahl, Travis McClure, and Robert O. Poyton*

The Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 This work supported by National Institutes of Health Grant GM30228 (to R.O.P), and a National Institutes of Health NRSA postdoctoral fellowship (to K.M.O.). ‡Present address: Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99775-7000 *To whom correspondence should be addressed. Tel.: 303-493-3823; Fax: 303-492-3883; Email: [email protected]

Running title: Intracellular distribution of flavohemoglobin in yeast

JBC Papers in Press. Published on December 20, 2004 as Manuscript M411478200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on August 20, 2019

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Summary

Yeast flavohemoglobin, YHb, encoded by the nuclear gene, YHB1, has been implicated in

both the oxidative and nitrosidative stress responses in Saccharomyces cerevisiae. Previous studies

have shown that expression of YHB1 is optimal in normoxic or hyperoxic conditions yet respiring

yeast cells have low levels of reduced YHb pigment, 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 among 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 can not be reduced in vivo or that YHb heme is maintained in an oxidized state in

respiring cells. By using an anti-YHb antibody, CO-difference spectroscopy, and by measuring NO

consumption we have found that YHb localizes to two distinct intracellular compartments, the

mitochondrial matrix and the cytosol, in respiring cells. 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.


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Introduction

Hemoglobins are found in all major groups of organisms, including plants, vertebrates,

invertebrates, protozoa, bacteria, and fungi (1). They make up a diverse super-family of proteins

that are grouped together because they all bind oxygen reversibly and they all possess a conserved

heme binding domain, the “myoglobin fold” (2). Aside from these common features hemoglobins

are otherwise divergent in structure and complexity. Indeed, while most vertebrate hemoglobins

consist of two types of polypeptide subunits that have single heme domains and form tetrameric

oligomers microbial, invertebrate, and protozoan hemoglobins are far more varied. Bacterial,

fungal, and protozoan hemoglobins fall into two general types: dimeric hemoproteins composed of

two single heme domain polypeptides, and monomeric flavohemoproteins containing a single heme

binding domain at its N-terminus and a C-terminal FAD-binding domain that is related to those

found in proteins in the ferredoxin-NADP+ reductase (FNR) family (3). From DNA sequence

analysis these flavohemoglobins appear to form a distinct subgroup within the hemoglobin family

(1,4).

The role of vertebrate and invertebrate hemoglobins in the transport of O2 and CO2 has been

known for some time. Recently, it has been discovered that the hemoglobins of both eucaryotes and

prokaryotes have additional functions relating to nitric oxide (NO). For example, in mammals

hemoglobin functions to transport NO to effectors that regulate blood flow and, therefore, the rate of

delivery of oxygen to tissue cells (5-8). In contrast, the function of Escherichia coli hemoglobin

(HMP) is to protect cells against nitrosidative stress brought about by NO or NO-donating S-

nitrosothiols (9-11). This hemoglobin functions in both aerobic and anaerobic cells. In the presence

of air it functions as an NO oxygenase that generates mainly nitrate, and in the absence of air it

functions as an NO-reductase that generates nitrous oxide. HMP may also play a role in oxidative

stress (9). The flavohemoglobin of Ralsonia eutropha is involved in denitrification, with the

production of N2O (12). The genes that encode the flavohemoglobins of R. eutropha, and Bacillus


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subtilis are up-regulated under oxygen limiting conditions (13, 14). Together, these findings suggest

a multiplicity of roles for hemoglobins.

The physiological role(s) of the flavohemoglobin found in S cerevisiae cells is (are) still

somewhat unclear. The expression of its gene, YHB1, is induced by oxygen, which is opposite to

the effects of oxygen on the expression of most bacterial hemoglobin genes. This has been taken to

indicate that the hemoglobins of yeast and bacteria may have different functions (15). Several

observations suggest physiological connections among the expression/ function of this protein,

mitochondrial respiration, and oxidative and nitrosidative stress. First, respiring S. cerevisiae cells

have low levels of reduced YHb pigment, detected by carbon monoxide (CO)- photolysis difference

spectroscopy (16). The intracellular levels of reduced YHb CO-photoproduct pigment are increased

in cells in which the mitochondrial respiratory chain has been compromised by either mutation (16)

or respiration inhibitors (17,18). Its level also increases in cells expressing the hypoxic isoform, Vb,

of cytochrome c oxidase subunit V under aerobic conditions (16). Second, yeast cells carrying a

deletion in YHB1, the structural gene for YHb become sensitive to some conditions that promote

oxidative (19) and other forms of stress (15). It has also been reported that some conditions

promoting oxidative stress increase the expression of YHB1 (19), but YHB1 expression under some

of these conditions have been questioned (15). Finally, it has been reported recently that YHb

metabolizes NO and functions as an NO oxygenase under aerobic conditions and an NO reductase

under anaerobic conditions (4).

To better understand the physiological function of YHb we have examined here the

relationship between respiration and the level of assembled YHb, have established the intracellular

location of YHb, and have looked at the effects of oxygen, respiration, oxidative stress, and the

mitochondrial genome on intracellular YHb localization.


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Experimental Procedures

Strains, Media, and Growth Conditions. The following strains of S. cerevisiae were

used: JM43 (MATα his4-580 trp1-289 leu2-3, 112 ura3-52 [rho+]) (20) ; JM43 ρ°, an

isochromosomal respiratory-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). For aerobic

growth, cells were grown on a shaker (200 r.p.m.) at 28 - 30 0C in YPGal (1% Difco yeast extract,

2% Difco Bacto-peptone, 2% galactose). For anaerobic growth, cells were grown in a fermenter

sparged with 2.5% CO2 in O2-free N2, as described (22). Yeast strains used for examining the

effects of mutations in CuZn or Mn superoxide dismutase on YHb were: EG103 (MATα, leu2-3,

his3∆1, trp1-289, ura3-52), EG110 (EG103 with sod2∆::TRP1), EG118 (EG103 with

sod1∆A::URA3), EG133 (EG103 with sod1∆A::URA3, sod2∆::TRP1) (23). They were kindly

provided by Edith Gralla. For these experiments cells were grown to mid-exponential phase in YPD

media (1% Bacto-yeast extract, 2% bacto-peptone, and 2% dextrose) in a shaker at 200 rpm and

28°C.

Preparation of Whole Cell Lysates. Whole cell extracts were prepared from cells grown to

mid-exponential phase in YPGal or YPD. Cells were harvested by centrifugation (5,000 g for 10

min), washed twice with ice-cold distilled water and re-suspended at 0.5 g wet weight cells per ml in

0.5 ml of RIPA buffer (0.2 M NaCl, 0.125 M NaPO4, pH7.0, 0.0125% (v/v) NP40, 0.125% SDS,

0.03 M Na deoxycholate) in a conical microfuge tube containing 1 g of glass beads (0.25-0.5 mm

diameter). Cells were broken by vortexing in two one minute intervals, separated by one minute on

ice. Cell debris was removed by centrifugation at 5000 X gmax in a Beckman Microfuge 18. The

supernatant was saved and frozen at -80oC until ready for use.

Isolation of Mitochondrial and Cytosol Fractions. For preparation of mitochondrial and

cytosolic fractions, cells were grown to mid-exponential phase, harvested, and spheroplasted as


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described (24). All subsequent steps were performed at 4o C. Spheroplasts were harvested by

centrifugation (5 min at 3,000 X g), washed gently in post-spheroplast buffer (1.5 M sorbitol, 1 mM

Na2EDTA, 0.1% BSA, pH 7.0), and sedimented at 3,000 X g for 5 min. Washed spheroplasts were

resuspended in lysis buffer (0.6 M mannitol, 2 mM Na2EDTA, 0.1% BSA, pH 7.4), lysed in a

Sorvall Omnimixer (Newton, CT) at low speed for 3 sec and at full speed for 45 seconds, and then

centrifuged for 5 min at 1,900 X g to pellet unbroken cells, nuclei, and debris. The resulting

supernatant was decanted and centrifuged for 10 min at 12,100 X g to pellet mitochondria. The

mitochondrial pellet was washed by resuspension in mitochondrial lysis buffer minus BSA (pH 7.0),

homogenized with a glass/teflon homogenizer, and centrifuged at 1,651 X g for 5 min. The

resulting supernatant was decanted and centrifuged at 23,500 X g for 10 min to pellet the

mitochondria. The post-mitochondrial supernatant, collected after the 23,500 X g centrifugation,

was used as the cytosolic fraction. Protein in the mitochondrial fractions from aerobically-grown

JM43 cells, anaerobically-grown JM43 cells, aerobically-grown JM43ρo cells, and aerobically-

grown JM43GD5ab cells accounts for 9.8%, 9%, 8.5%, and 9.8%, respectively, of the combined

protein in mitochondrial and cytosolic fractions.

Sub-fractionation of Mitochondria. Mitochondrial subfractions were prepared by a

modification of the digitonin fractionation procedure described (25). In this procedure, freshly

isolated mitochondria are converted to mitoplasts, during which the outer membrane and inter-

membrane space fractions are removed. The mitoplasts are then disrupted and the inner membrane

and matrix fractions are separated. To prepare mitoplasts, mitochondria were suspended at a protein

concentration of 10 mg per ml of mitoplast suspension buffer (0.6 M mannitol, 10 mM NaPO4, pH

7.0) and a 1 ml portion of the suspension was treated for 1 min on ice with 0.35 mg digitonin per mg

mitochondrial protein. All subsequent steps were performed at 4o C. The suspension was diluted

immediately with 5 volumes of mitoplast suspension buffer and centrifuged for 10 min at 12,000 x

gmax. The pellet is the mitoplast fraction. The supernatant was carefully separated from the pellet

and centrifuged for 60 min at 144,000 x gmax. The resulting pellet and the supernatant were used as

the outer mitochondrial membrane and the intermembrane space fractions, respectively. The inner


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membrane and matrix fractions were prepared from the mitoplasts, collected above, as follows.

Mitoplasts were resuspended at 10 mg of protein per ml in 100mM NaPO4, pH 7.0, sonicated for 20

sec at 50 watts with a Branson Sonifier (Model W 185) equipped with a microtip. Sonicated

mitoplasts were centrifuged for 20 min at 95,000 X gmax. The pellet is the inner membrane fraction

and the supernatant is the matrix fraction. The pellet was washed by resuspension in 100mM

NaPO4, pH 7.0 and recovered by centrifugation at 95,000 X gmax for 20 minutes.

Partial Purification of Cytosolic YHb. Freshly isolated cytosolic fraction was subjected a

series of ammonium sulfate fractionations, as modified from Mok et al. 1969 (26). Briefly,

ammonium sulfate was added to 30% saturation, the sample incubated at 40C for 15 min with

stirring, and then centrifuged at 27,000 x gmax for 15 min at 4°C. The pellet was suspended in 100

mM NaPO4, pH 7.0 and saved. The supernatant was adjusted to 45% ammonium sulfate, the

suspension was incubated at 40C for 15 min with stirring, and then centrifuged at 27,000 x gmax for

15 min at 4°C. The pellet was suspended in 100 mM NaPO4, pH 7.0 and saved. The supernatant

was adjusted to 65% ammonium sulfate, the suspension was incubated at 40C for 15 min with

stirring, and then centrifuged at 27,000 x gmax for 15 min at 4°C. The pellet was suspended in 100

mM NaPO4, pH 7.0 and saved. The supernatant was adjusted to 95% saturation, incubated at 40C

for 15 min with stirring, and then centrifuged at 27,000 x gmax for 15 min at 4°C. The pellet was

suspended in 100 mM NaPO4, pH 7.0 and saved. The remaining supernatant was saved. All pellets

were re-suspended to a volume of 3 ml per 100 ml starting volume. All fractions were stored at 4°C

until spectra were recorded and then frozen at -70°C for immunoblot analysis.

Difference Spectroscopy. Whole cell suspensions were prepared for CO-photolysis spectra

as follows. A concentrated cell suspension (0.45 g wet weight /ml) was adjusted to 1 % (w/v)

glucose in a sealed small test tube and incubated without agitation for 5 min at room temperature to

achieve anaerobiosis. The suspension of reduced cells was then adjusted to 30% ethylene glycol

and bubbled with CO for 10 min, prior to transfer to a cuvette. The cuvette was frozen in liquid

nitrogen and CO-photolysis difference spectra on the frozen samples recorded as described (16).

Reduced, carbon monoxide (CO)-ligated difference spectra of cell fractions were recorded at room


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temperature, as follows. Each cell fraction was reduced with sodium dithionite and a spectrum was

recorded. Then each was bubbled slowly with CO gas (99.5%) for 5 min. The visible/Soret spectra

of the same sample were recorded before and after exposure to CO. The base line spectrum,

obtained from the difference between the two spectra of the same reduced sample, was subtracted

from the reduced minus CO-ligated difference spectra. All spectra were recorded using an SLM

AMINCO DW2000 dual wavelength scanning UV-visible spectrophotometer (SLM Instruments,

Inc.) with the following settings: dual beam, wavelength acquisition, filter slow, slit width 0.8 nm,

recording monochrometer (monochrometer 2) 1 from 390 nm to 700 nm, and the reference

monochromator (monochrometer 1) set to a wavelength of 577nm.

Preparation of Antibodies. Polyclonal antibodies were prepared to YHb, PET100p and

cytochrome c oxidase subunit IV. For YHb, an antibody was obtained against a peptide synthesized

to correspond to amino acid residues 379 to 397 (17) and for PET100p an antibody was obtained

against a peptide synthesized to correspond to residues 95 to 111 (27). They were synthesized with

an amino terminal cysteine and coupled through this cysteine to Maleimide Activated Keyhole

Limpet Hemacyanin. The peptide-KLH conjugate was mixed with Freunds Complete Adjuvant and

1 mg of conjugate was injected subcutaneously into New Zealand white rabbits. Booster injections

(0.5 mg) were given with Freund's Incomplete Adjuvant at 2 and 8 weeks. Antiserum was collected

at 2 week intervals with the terminal collection at 12 days after the final boost. Antibodies to HPLC

purified cytochrome c oxidase subunit IV were prepared as described (28). Monoclonal antibodies

to yeast phosphoglycerate kinase (and porin) were obtained from Molecular Probes, Inc. (Eugene,

OR).

Measurement of NO Consumption. NO consumption was measured using a Clark-type NO

electrode (World Precision Instruments, Sarasota, FL). All measurements were done in triplicate in a

water jacketed chamber with stirring, at 30°C. The electrode was calibrated with a 50µM solution of

KNO2 to generate a series of known concentrations of NO (100, 200, 400 nM) in 10 ml of a 0.1M

KI, 0.1 M H2SO4 solution. NO consumption of whole cell extracts, mitochondrial or cytosolic

fractions was then measured in 2mls of PBS (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl),


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250 µM NADH, and 0.1mM EDTA after the addition of 2µM NO from an NO-saturated solution of

distilled water. Preparation of the NO-saturated solution was as described by the manufacturer of

the NO electrode. Using a buffer control it is possible to determine the amount of NO injected into

the chamber as well as its stability in the absence of cell extract.

Northern Blotting. For Northern analysis, RNA samples (30 µg) were separated on 1.5%

agarose gels containing 0.22M formaldehyde (29), transferred to Scleicher and Schuell Nytran

membranes, and hybridized according to the manufacturer's suggestions. DNA probes were

prepared by random-primer labelling of double stranded DNA fragments using [α-32P] dCTP .

Probes were a 600-bp Ssp1 fragment for YHB1 and a 500-bp Sty1 fragment for ACT1. Blots were

quantitated with an Ambis Radioanalytic Imaging system.

SDS-PAGE Electrophoresis and Immunoblot Analysis. SDS-PAGE were performed either

on 16% SDS polyacrylamide gels containing 10% glycerol and 3.6 M urea (24) or 10% SDS

polyacrylamide gels (30), as indicated in the Figure Legends. Following electrophoresis, proteins

were electroblotted to nitrocellulose and immunoblots were quantitated from multiple exposures

using KODAK 1D image analysis software.

Miscellaneous Methods. Protein determination was performed using either the Lowry assay

(31) or the BCA assay (Pierce Biotechnology) with BSA as a standard.


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Results

Expression of YHb in rho+ and rhoo cells. Previously, we have reported that the

level of the reduced YHb CO-photoproduct is low in respiration-proficient cells and is

increased in respiration-deficient mutants (16). This can be seen in Figure 1, which

compares the CO photolysis difference spectra (CO-reduced minus reduced) of whole

suspensions of JM43, a respiratory-proficient rho+ strain, and its isochromosomal derivative,

JM43ρo, a respiratory-deficient rho0 strain. The major CO- liganded pigment in JM43 is

cytochrome c oxidase. It absorption maxima are at 447 and 610 nm and absorption minima

at 434 and 590 nm. In contrast, the main CO- liganded pigment in JM43ρo is YHb. Its

absorption maxima are at 441, 557 and 590 nm with absorption minima at 423, 538 and 574

nm. From Figure 1 and from previous studies that have examined the CO- recombination

kinetics of JM43 (16) it is clear that JM43 lacks a spectrally detectable CO-binding pigment

that has the absorption characteristics of YHb. In contrast, JM43 ρo cells contain a CO-

binding pigment with all of the spectral signatures of YHb. Despite the absence of a CO-

spectral signature assignable to YHb in JM43 cells we have found that YHB1 mRNA levels

in JM43 and JM43ρo are essentially identical (Figure 1B).

The presence of YHb mRNA but absence of a CO-liganded YHb in JM43 cells

suggested one of three things. First, JM43 cells may not translate YHB1 mRNA and hence

lack the YHb apo-protein. Second, JM43 cells may lack assembled and functional YHb.

Third, JM43 cells may express and assemble YHb but maintain the heme of YHb in an

oxidized state, which is incapable of binding CO. In order to examine these possibilities we

first asked if the YHb apoprotein is present in rho+ cells. A polyclonal antibody, anti-YHbC,

to a 19 amino acid synthetic peptide whose sequence corresponds to that predicted from the

carboxyl terminus of the YHB1 gene (Figure 2A), was produced to address this question.

The reactivity and specificity of this antibody was analyzed by immuno-blot analysis of

whole cell extracts prepared from JM43 and DR11, a derivative of JM43 that carries a null

mutation in the YHB1 gene. From Figure 2B it is clear that the antibody detects an antigen


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in whole cell extracts from JM43 but not from DR11. Th is antigen has an apparent

molecular weight of 47,000 Da, which is consistent with the molecular weight predicted for

YHb from the sequence of its gene (17). Because the antigen recognized by anti-YHbC is

present in JM43 but not DR11 and has a size similar to that predicted from the DNA

sequence of YHB1 we conclude that anti-YHbC recognizes and is specific for YHb. We also

conclude that YHb apo-protein is expressed in JM43 cells. To compare levels of YHb

apoprotein in JM43 and JM43ρo cells we subjected whole cell extracts from both to

immuno-blot analysis. From Figure 2C it can be seen that YHb levels in JM43ρ0 cells are

57% of the level in JM43 cells. The finding that YHb protein is present in JM43 and at

higher levels than in JM43ρo allows us to conclude that the absence of a spectrally detectable

YHb protein in JM43 cells can not be explained by a low level of expression of the YHb

apoprotein.

Assembled YHb is Present in Both the Cytosol and Mitochondrial Matrix of

Respiration-Proficient Cells. The results presented above make clear that the YHb apo-

protein is expressed in JM43 cells even though no CO-difference spectral signature for

assembled YHb can be detected in these cells. To gain further insight concerning this

apparent discrepancy we examined the intracellular location of YHb. We were particularly

interested in determining whether YHb is a mitochondrial protein because rhoo cells lack

both a mitochondrial genome and mitochondrial respiration. By immuno-blotting cellular

fractions against antisera to YHb, Pet100p (an inner mitochondrial membrane protein), and

3- phosphoglycerate kinase (a cytosolic protein) we find that YHb is present in both

mitochondrial and cytosolic fractions (Figure 3A) while, as expected, PET100p is present

only in the mitochondrial fraction, and PGK is present mainly in the cytosolic fraction.

Immunoblot analysis reveals the complete lack of detectable YHb in both the cytosolic and

mitochondrial fractions (Figure 3B) from strain DR11, indicating that the disruption of the

YHB1 gene alone is sufficient to abolish both cytosolic and mitochondrial forms of YHb.


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To localize YHb within mitochondria we prepared mitochondrial sub-fractions and

immuno-blotted them against antisera to YHb, Pet100p, and COX IV (subunit IV of

cytochrome c oxidase). Pet100p is an integral inner membrane protein while COX IV is a

peripheral protein subunit that binds holo-cytochrome c oxidase on the matrix side of the

inner membrane (32, 33). The assembled form of COX IV is located to the inner membrane

while the unassembled form is located in the matrix (34). From Figure 4 it can be seen that

YHb is present in mitoplasts (Lane 2), which contain both the inner membrane and matrix.

It is not present in the inter-membrane space (lane 5) or outer membrane (lane 6). Upon

fractionating mitoplasts into their two sub-fractions, the inner membrane (lane 3) and matrix

(lane 4), we find that most of the YHb present in mitochondria and mitoplasts resides in the

mitochondrial matrix. As expected for a matrix located protein mitochondrial YHb is

resistant to proteolysis by externally added trypsin (data not shown) Further evidence for

the presence of YHb in the mitochondrial matrix comes from CO-difference spectra of this

fraction (Figure 5). Both whole mitochondria (panel A) and the inner mitochondrial

membrane fraction (panel B) exhibit a major absorption maxima at 447 nm, the CO-spectral

signature of cytochrome c oxidase. In contrast, the mitochondrial matrix fraction (Panel C)

exhibits a major absorption maxima at 441 nm, the CO-spectral signature of YHb. It is

interesting that YHb is not observed by CO-difference spectroscopy of intact mitochondria

but is observed when the matrix fraction derived from intact mitochondria is assayed.

To ask if the cytosolic YHb antigen has an YHb CO-spectral signature we subjected

the cytosol to ammonium sulfate fractionation and performed immuno-blots and CO-

difference spectroscopy on the fractions obtained. From Figure 6A it can be seen that the

YHb antigen is not precipitated until the ammonium sulfate concentration exceeds 45%. It is

most abundant in fraction 4, which comes down between 45 and 65% ammonium sulfate.

CO-difference spectral analysis of this fraction as well as un-fractionated cytosol reveals that

each contains a pigment with the spectral signature of YHb. These findings permit three

conclusions. First, the YHb antigen and the pigment with a CO-spectral signature co-purify.


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Second, the YHb antigen present in the cytosolic fraction is assembled with its prosthetic

groups. And third, even though the YHb pigment can not be observed in whole cells it is

nonetheless present and is easily observed in broken cell extracts.

YHb is Active in NO Consumption in Both Mitochondria and Cytosol. Previously,

it has been shown that yeast cell lysates consume NO and that this is attributable to YHb (4).

We have been able to confirm this here by assaying whole cell extracts from JM43 and

DR11 for their ability to consume NO (Figure 7A). In these studies, NO is injected into an

air tight chamber and NO consumption is followed with an NO electrode. Using a buffer

control it is possible to determine the amount of NO injected into the chamber as well as its

stability in the absence of cell extract. The buffer control trace and that from the DR11

extract are similar (Figure 7A), indicating that the disruption of the YHB1 gene nearly

completely eliminates NO consumption in whole cell extracts. In contrast, NO consumption

in a comparable amount of JM43 whole cell extract is extremely rapid. Indeed, it is so rapid

that the initial peak of added NO observed in the buffer control is absent and can not be

observed until the amount of whole cell extract added is reduced 10-fold (data not shown).

From Figures 7B and 7C it can be seen that both mitochondrial and cytosolic fractions from

JM43 but not DR11 are also capable of consuming added NO. The initial peak of NO

observed in the buffer control is greatly diminished when JM43 mitochondrial protein or

cytosolic protein is assayed. These findings clearly establish that the YHb proteins in both

the mitochondrial matrix and cytosol fractions are capable of NO consumption.

YHb Levels in sod1 and sod2 Mutants. The localization of functional YHb to both

mitochondrial and cytosolic fractions is surprising and led us to ask if the relative levels of

YHb in these two compartments is regulated. To address this we first examined the

intracellular distribution in cells grown under conditions known to regulate the expression of

YHB1. First, we examined the intracellular location of YHb in mutants defective in one or

both forms of superoxide dismutase. Previously, we have reported that the YHB1 transcript

levels are increased nearly three-fold in a strain lacking both CuZn superoxide dismutase


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(encoded by SOD1) and Mn superoxide dismutase (encoded by SOD2) (19). Immunoblots

of whole cells extracts from sod1, sod2 and sod1sod2 mutants have shown that YHb protein

levels are increased in sod1 or sod1sod2 mutants but are unaffected in a sod2 mutant. From

Figure 8A it is clear that YHb concentrations are increased in both mitochondrial (Lane 3,4)

and cytosolic fractions (Lane 7,8) from sod1 or sod1sod2 mutants. Mitochondrial YHb

concentrations in these mutants are approximately three times higher than in the wild-type.

Cytosolic YHb concentrations are approximately five-fold higher in sod1 mutants and seven-

fold higher in the sod1sod2 mutant, compared to the wild-type. In contrast, YHb

concentrations in mitochondrial (Lanes 1,2) and cytosolic fractions (Lanes 5,6) are

equivalent in the wild-type and sod2 strains. These findings indicate that increased

expression of YHb observed in a sod1sod2 mutant is brought about by the lack of CuZn

superoxide dismutase but not Mn superoxide dismutase, and that in the absence of CuZn

superoxide dismutase YHb levels increase in both the mitochondrion and cytosol.

Intracellular Distribution of YHb in rhoo, nuclear pet and anoxic cells. To

examine further the relationship between respiration and YHb we have determined total

levels of YHb in mitochondrial and cytosolic fractions from JM43ρo cells, JM43GD5ab, and

JM43 cells grown under anoxic and normoxic conditions. These three strains were chosen to

examine the effects of respiration, the mitochondrial genome, and the presence of oxygen on

the intracellular localization of YHb. They are all iso-chromosomal. JM43ρo differs from

JM43 in lacking both a mitochondrial genome and the mitochondrially-encoded proteins of

the respiratory chain. JM43GD5ab differs from JM43 in lacking cytochrome c oxidase and,

hence, respiration. It contains a wild-type mitochondrial genome. Anoxic JM43 cells also

retain a mitochondrial genome and the components of the respiratory chain (David et al.

unpublished) but differ from normoxic JM43 cells in being grown in the absence of oxygen,

the terminal electron acceptor of the respiratory chain.

Because YHb is present only in the mitochondrial and cytosolic fractions it is

possible to determine the relative total content of YHb in each fraction from the relative


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concentration of YHb in each fraction and the relative total protein content of each fraction

(Table 1). The majority of YHb in aerobically grown JM43 cells is present in the cytosolic

fraction; this represents 36.3 arbitrary units of the protein. As seen above (Figure 1 and

Table 1), the level of YHb in JM43ρo cells is about 57 % of its level in JM43 cells.

Surprisingly, nearly all of the YHb protein in JM43ρo cells is found in the cytosol. (Figure

8B and Table 1). JM43ρo cells differ from JM43 cells both genotypically and

phenotypically; they lack a mitochondrial genome and they lack respiration. To address

whether the intracellular distribution of YHb is affected by the lack of the mitochondrial

genome or by the lack of respiration we examined mitochondrial and cytosolic YHb levels in

JM43GD5ab, a nuclear pet mutant that retains a wild type mitochondrial genome but carries

gene disruptions in both subunit V isoforms of cytochrome c oxidase and does not respire.

This strain distributes its YHb between mitochondrial and cytosolic fractions (Figure 8, lanes

5 and 6; Table 1) in the same way as JM43, its wild type parent. Hence, the altered

distribution of YHb in JM43ρo can not be attributed to the lack of respiration per se. Further

evidence for this comes from an examination of the intracellular distribution of YHb in

anoxically-grown JM43 cells. Anoxic growth has only a slight effect on total YHb protein

levels in JM43 cells (Table 1). Interestingly, however, it does affect the intracellular

distribution of YHb in a way that is opposite from that observed in rhoo cells. All of the YHb

in anoxic JM43 cells is located in the pro-mitochondrial fraction (Figure 8, lanes 7 and 8

Table 1). None can be observed in the cytosol. Interestingly, the number of arbitrary units

of YHb protein in promitochondria of anoxic JM43 cells is nearly identical to the number of

arbitrary units of units of YHb protein in the cytosolic fraction of aerobic JM43 cells.

When considered together, these findings suggest that the altered distribution of YHb

observed in rhoo cells is the result of the lack of the mitochondrial genome and not the lack

of respiration. These results and those obtained with the sod mutants, indicate that yeast

cells can differentially regulate the intracellular level of YHb and its intracellular distribution

between cytosolic and mitochondrial fractions.


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Discussion

This study presents three novel findings concerning the YHb flavohemoglobin in yeast.

First, although CO-liganded YHb is not detectable in intact reduced rho+ cells it is detectable in cell

fractions from rho+ cells. Second, functional YHb localizes to two distinct intracellular

compartments: the cytosol and mitochondrial matrix. And third, the distribution of YHb between

the cytosol and mitochondrial matrix is affected by anoxia and the mitochondrial genome.

Our findings that YHB1 mRNA and YHb protein are expressed in Rho+ cells, and that a CO-

liganded YHb is not detectable in intact rho+ cells but is detectable in cell fractions from rho+ cells

seem to be paradoxical. There appear to be at least two plausible explanations. One explanation for

this apparent discrepancy is that accessibility of intracellular YHb to NAD(P)H or NADH, its

physiological reductants (35), is limited in vivo, most likely due to competition between YHb and

the mitochondrial respiratory chain. In order to bind CO the heme group in YHb must be reduced.

In the absence of reduced heme, CO can not bind to YHb in vivo and a spectral signature would not

be observed. According to this explanation, CO-liganded YHb would be observable in cell fractions

and in rhoo cells because the competition for NAD(P)H and/or NADH would be absent. A second,

and more likely, explanation is that YHb heme is maintained in the oxidized state in intact cells

because it has a high turnover rate with NO (35). As such, electrons would be released from the

heme group and act on NO as soon as it binds to the heme. According to this explanation, CO-

liganded YHb should be observed when levels of its substrate, NO, decrease. Because NO is very

unstable its level should decrease dramatically when cells are broken. This decreased level of NO in

broken cell extracts could explain why CO-liganded YHb is observed in cell fractions from rho+

cells but not in intact rho+ cells. Because CO-liganded YHb is observed in rhoo cells, this

explanation would also imply that the respiratory chain and/or the mitochondrial genome is required

for NO production. Involvement of the mitochondrial respiratory chain in NO production could

explain our observation that a CO-liganded YHb pigment can not be observed in intact respiring

mitochondria but can be observed when the mitochondrial matrix sub-fraction is isolated.


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The presence of YHb that is functional in NO consumption in both cytosolic and

mitochondrial fractions of JM43 implies that NO is produced in both cellular compartments in yeast

cells. This is not surprising given a recent report that yeast cells express proteins that cross- react

with all three mammalian nitric oxide synthase isoforms – neuronal (NOS1), inducible (NOS2) and

endothelial (NOS3)(36). Although the intracellular location of the yeast isoforms has not been

established yet at least one of the mammlian nitric oxide isoforms (NOS3) is associated with

mitochondria (37, 38).

Localization of YHb to mitochondria can help explain how it functions both in oxidative

stress (19) and in nitrosidative stress (4). Indeed, it is likely that YHb protects yeast cells from

oxidative stress primarily by controlling levels of mitochondrial NO, which is a potent inhibitor of

cytochrome c oxidase and respiration (39) and a reactant that together with superoxide can form

peroxynitrite (40). Peroxynitrite participates in some forms of protein nitration (41). In the absence

of YHb, NO levels would increase (4) and inhibit cytochrome c oxidase, leading to the production

of superoxide, and hence enhanced levels of peroxynitrite. Thus, YHb can reduce superoxide levels

not by dismuting superoxide, like superoxide dismutase, but rather by reducing its formation via the

respiratory chain. Our finding that YHb is present mainly in the cytosol of rhoo cells is consistent

with this hypothesis because these cells lack important components of the mitochondrial respiratory

chain (cyochrome bc1 and cytochrome c oxidase) and hence are respiration-deficient and incapable

of producing superoxide. Our finding that YHb is present in both the cytosol and mitochondrion of

JM43GD5ab cells is also interesting because although these cells lack cytochrome c oxidase they

still retain the bc1 complex and a partial respiratory chain. Hence, they are capable of superoxide

production. The presence of YHb in the promitochondria of anoxic cells is somewhat surprising

considering that these cells are grown in the absence of oxygen and have greatly reduced levels of

oxidative stress (42). However, this finding implies that NO is present in anoxic mitochondria and

is consistent with recent studies that have demonstrated that the concentration of NO increases in rat

liver mitochondrial suspensions subjected to low oxygen tensions (43) and that mitochondrial

protein S-nitrosylation occurs in anoxic rat liver mitochondria (44).


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Our finding that the ratio of YHb in mitochondrial and cytosolic fractions differs in rho+,

rho0, and anoxic cells indicates that yeast cells can differentially regulate the intracellular

distribution of YHb between cytosol and mitochondrion. Precedence for the regulated differential

targeting of a single yeast protein between two different intracellular locations comes from studies

on the dual targeting of catalase A to mitochondria and peroxisomes (45). The level of respiration

and/or growth on oleic acid influences the intracellular distribution of this protein. The presence or

absence of respiration does not appear to influence the intracellular distribution of YHb because the

mitochondrial/cytosolic ratio of YHb is identical in JM43, a respiration-proficient strain, and

JM43GD5ab, a respiratory-deficient derivative of JM43. Moreover, the ratios of

mitochondrial/cytosolic YHb in JM43ρo and anoxic JM43 cells are nearly the converse of one

another even though both cells are respiration-deficient. These findings clearly demonstrate that the

absence of respiration per se is not a determinant of the intracellular localization of YHb, and

indicate that anoxia and the mitochondrial genome influence the intracellular localization of YHb.

Previously, we have shown that the mitochondrial genome, acting independently of

respiration and oxidative phosphorylation, affects expression of a subset of nuclear genes (46) and

have termed this type of mitochondrial-nuclear crosstalk Intergenomic signaling. Genes that are

regulated by Intergenomic signaling are specifically down-regulated in cells that lack a

mitochondrial genome (46). More recently, using microarray analysis, we have been able to show

that this pathway affects the expression of over 100 nuclear genes in yeast (Dong, Liang, and

Poyton, unpublished). The target genes of this pathway are different than the target genes of the

better understood Retrograde Regulation pathway, which involves mitochondrial respiration and

which connects altered mitochondrial respiration to altered carbon and nitrogen metabolism (47). In

considering how the mitochondrial genome affects the intracellular distribution of YHb is seems

likely that one of the nuclear target genes affected by Intergenomic signaling encodes a protein that

functions in the intracellular partitioning of YHb.

Two lines of evidence indicate that the mitochondrial and cytosolic forms of YHb are

derived from a single nuclear gene, YHB1. First, a null mutation in YHB1 simultaneously abolishes


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both proteins. Second, transformation of a yhb1 null mutant with a plasmid containing the YHb

protein leads to the appearance of YHb in both the mitochondrial matrix and the cytosol (19,

Fontaine and Poyton, unpublished observations). The presence of a single nuclear gene product in

multiple intracellular compartments is not without precedence. Indeed, a growing number of

mitochondrial proteins are being found in both the cytosol and mitochondrion (48, 49). These

include the histidyl-tRNA synthase (50), fumarase (51, 52), CuZn superoxide dismutase (53), and

dihydroxybutanone phosphate synthase (54) from yeast, thioredoxin from Drosophila (55) , the

thioredoxin glutathione reductase from Echinococcus (56), and several human proteins (48),

including aspartate aminotransferase (57) and the Wilson’s copper transport protein (58). Close

examination of several of these proteins has revealed a multiplicity of mechanisms for the

localization of proteins to the mitochondrion and cytosol (48). These include the use of multiple

transcription start sites, multiple translation start sites, and mitochondrial import followed by

retrograde movement out into the cytosol (52). Currently, it is not clear which of these mechanisms

is used by YHb. Moreover, because the DNA sequence of YHB1 does not predict the presence of a

mitochondrial signal peptide it is also not clear how YHb is targeted to the mitochondrial matrix.

One possibility is that, like some other nuclear-coded mitochondrial proteins, YHb uses an internal

targeting sequence (59, 60) for import into the mitochondrion. Alternatively, the YHb gene may

produce an alternative transcript that puts a mitochondrial targeting sequence on the YHb protein.

These possibilities are currently under study.

Acknowledgements We gratefully acknowledge the technical assistance provided by Susan Fontaine.


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Table 1. Effects of Respiration, Anoxia and the Mitochondrial Genome on Intracellular Levels and Distribution of YHb Between Mitochondrial and Cytosolic Fractions

Strain Culture condition

Whole cell levela

Total unitsb in: % of total in:

Mitochondriaa Cytosola Mitochondria Cytosol

JM43 aerobic 100 5.9 36.3 13.9 86.1 JM43 anaerobic 90.5 38.1 0 100 0 JM43ρo aerobic 56.7 0.1 23.8 0.4 99.6 JM43GD5ab aerobic 88.0 5.5 35.8 13.3 86.7

aValues given are the average of at least two determinations of YHb levels in mitochondrial and cytosolic fractions.

b Total units are determined relative to those in JM43 cells grown under aerobic conditions, which is taken as 100%. Levels in JM43 grown under aerobic conditions are calculated from the percent concentration in mitochondrial and cytosolic fractions (determined by quantitating Western immuno-blots like those in Figure 8B) multiplied by the ratio of total protein accounted for by each fraction (see Experimental Procedures). Total units in mitochondrial and cytosolic fractions from JM43 cells grown under anaerobic conditions, JM43ρo cells, and JM43GD5ab cells are calculated as described above, and are normalized to their whole cell level.


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FIGURE LEGENDS

Figure 1. Expression of YHb in rho+ and rho0 cells. Panel A: Low temperature CO

photolysis difference spectra of strains JM43 and JM43ρo. Glucose reduced whole cell suspensions

( 0.8 g/ml wet weight) were bubbled for 10 min with CO, frozen in liquid nitrogen, placed in a pre-

cooled cryostat, equilibrated to -120oC, and a spectrum was recorded. They were then subjected to

photolysis and a second spectrum recorded. The pre-photolysis CO ligated base-line spectrum is

subtracted from that of the photoproduct. Panel B: Northern blot analysis of total RNA isolated

from JM43 (Lane 1) and JM43ρo (Lane 2). Total RNA was prepared from both strains and blotted

with both YHB1 and ACT1 probes as described under "Experimental Procedures". The YHB1/ACT1

ratios were determined by quantitation with a phosphorimager.

Figure 2. Generation and specificity of anti-YHbC and Comparison of YHb levels in rho+

and rho0 cells. Panel A: A polyclonal antibody was made to a 19 amino acid synthetic peptide

whose sequence corresponds to amino acids 379 to 397 of YHb. Panel B: Whole cell extracts were

prepared from strains JM43, a strain with a wild type YHB1 gene, and DR11, which carries a yhb1

null mutation. An aliquot of each was subjected to SDS-PAGE (16% gels) and immuno-blotted

with anti-YHbC. Lane 1, 10 µg of total cell protein from JM43; lane 2, 10 µg of total cell protein

from DR11. The migration positions of molecular weight markers, given in kDa, are shown at the

left. Panel C: Aliquots of whole cell extracts from JM43 and JM43ρo were analyzed on 16% SDS-

PAGE gels and immuno-blotted with anti-YHbC. Lane 1, 5 µg of total cell protein from JM43; lane

2, 5 µg of total cell protein from JM43ρo.

Figure 3. YHb is Present in Both Mitochondria and the Cytosol. Panel A: Immunoblot

analysis of cell fractions from JM43. Cell fractions were isolated from strain JM43, solubilized in

SDS, and subjected to SDS-PAGE (16% gels), as described in Experimental Procedures. After

SDS-PAGE the gels were blotted to nitrocellulose and detected with anti-YHbC (top panel), an

antibody to the inner mitochondrial membrane protein PET100p (middle panel), and an antibody the


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cytosolic protein 3- phosphoglycerate kinase (bottom panel). Lane 1, 10 µg of whole cell protein;

lane 2, 10 µg of cytosolic protein; lane 3, 10 µg of mitochondrial protein. Panel B: Immunoblot

analysis of mitochondria and cytosol from DR11. 10 µg of mitochondrial or cytosolic protein from

JM43 and DR11 were subjected to SDS-PAGE (16% gels) and then immunoblotted against anti-

YHbC. Lane 1, JM43 mitochondria; lane 2, JM43 cytosol; lane 3, DR11 mitochondria; lane 4,

DR11 cytosol.

Figure 4. Mitochondrial YHb Resides in the Matrix of Rho+ Cells. Mitochondrial

subfractions from strain JM43 were subjected to SDS-PAGE (16% gels) and immuno-blotted with

anti-YHbC, an antibody to Pet100p, and an antibody to cytochrome c oxidase subunit IV (COXIV).

Lane 1, whole mitochondria; Lane 2, mitoplasts; Lane 3, inner membrane; Lane 4, matrix; Lane 5,

outer membrane; Lane 6, intermembrane space.

Figure 5. CO-ligated Difference Spectra of Isolated Mitochondria, Mitochondrial Inner

Membrane, and Matrix Fractions. CO-ligated difference spectra of aliquots (1.7 mg protein per

ml) of whole mitochondria (Panel A), mitochondrial inner membrane (Panel B), and mitochondrial

matrix (Panel C) from JM43. Each fraction was reduced with sodium dithionite and a spectrum was

recorded. Then each was bubbled slowly with CO gas for 5 min. The visible/Soret spectra of the

same sample were recorded before and after exposure to CO. The base line spectrum, obtained from

the difference between the two spectra of the same reduced sample, was subtracted from the reduced

minus CO-ligated difference spectra.

Figure 6. CO-Ligated Difference Spectra of Unfractionated Cytosol and Partially Purified

Cytosolic YHb. The cytosolic fraction from JM43 was subjected to a series of sequential ammonium

sulfate precipitations (30%, 45%, 65%, and 95%) in order to partially purify the YHb protein.

Purification was followed by SDS-PAGE (16% gel) and immuno-blot analysis using anti-YHbC and

CO-difference spectroscopy. Panel A: Immuno-blot analysis of total cytosol (Lane 1), 30 %

ammonium sulfate precipitate (Lane 2), 45% ammonium sulfate precipitate (Lane 3), 65%


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ammonium sulfate precipitate (Lane 4), 95% ammonium sulfate precipitate (Lane 5), and the

supernatant that was removed from the 95% precipitate (Lane 6). Each ammonium sulfate

precipitate was re-suspended in 100 mM NaPO4, pH 7.0. An aliquot containing 10 µg protein was

loaded onto each lane. Panel B: CO-ligated difference spectra of aliquots (4 mg protein per ml) of

the unfractionated cytosol and the resuspended 65% ammonium sulfate precipitate. Spectra were

obtained as described in the legend to Figure 5. Top: cytosol. Bottom: resuspended 65% ammonium

sulfate precipitate.

Figure 7. NO consumption by whole cell lysates, mitochondrial, and cytosolic fractions

from JM43 and DR11. NO consumption was monitored with an NO electrode in an air tight

chamber. Reactions in whole cell lysates (Panel A), mitochondria (Panel B), and cytosol (Panel C)

from JM43 (solid line) and DR11 (light line) were initiated by injection of an NO-saturated solution

to a final concentration of 2 µM NO. The reaction chamber contained 200 µg of protein for each

sample and a buffer control (thick line) was included with each set of analyses.

Figure 8. Intracellular distribution of YHb in sod mutants and non-respiring cells. Cells

were grown to mid-logarithmic phase in YPD media at 28°C and 200 rpm. Cells were harvested and

mitochondrial and cytosolic compartments isolated as described in Experimental Procedures. Panel

A: Immuno- blot analysis of YHb in mitochondrial and cytosolic fractions from sod mutants. 10 ìg

of protein per lane was separated on a 10% SDS-PAGE gel and immuno-blotted with anti-YHbC.

Lanes 1-4 ; mitochondrial fractions from EG103 (wild type), (lane 1), EG110 (sod2 mutant )(lane

2) EG118 (sod1 mutant)(lane 3), and EG133 (sod1sod2 mutant), (lane 4). Lanes 5-8; cytosolic

fractions from EG103 (wild type) (lane 5), EG110 (sod2 mutant) (lane 6), EG118 (sod1 mutant)

(lane 7), and EG133 (sod1sod2 mutant) (lane 8). Panel B. Immuno-blot analysis of YHb in non-

respiring cells. 10 µg protein was loaded per lane, separated by SDS-PAGE and immunoblotted

with anti-YHbC. Lane 1; cytosol from aerobically- grown JM43: Lane 2; mitochondria from

aerobically-grown JM43: Lane 3; cytosol from JM43ρo; Lane 4; mitochondria from JM43 ρo; Lane


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5; cytosol from JM43GD5ab: Lane 6; mitochondria from JM43GD5ab: Lane 7; cytosol from

anaerobically-grown JM43; Lane 8; promitochondria from anaerobically-grown JM43


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1.0 0.57


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1 2 3

YHb

Pet100p

PGK

A

1 2 3 4B

YHb


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1 2 3 4 5 6

YHb

PET100

COX IV


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500 600 700400

Abs

orba

nce

Wavelength (nm)

0.002

0.002

0.002

441

423

A

B

C


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1 2 3 5 64A

B

441

423

400 500 600 700

0.005

0.005

Wavelength (nm)

Abs

orba

nce

590557

572

557 590

574


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2500

2000

1500

1000

500

0

2500

2000

1500

1000

500

0

3000

2500

2000

1500

1000

500

0

NO

(A

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ary

Uni

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0 10 20 30 40 50 60 70Time (sec)

Buffer Control

DR11

JM43

Buffer Control

DR11

JM43

Buffer Control

DR11

JM43

A

B

C


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Nina Cassanova, Kristin M. O'Brien, Brett T. Stahl, Travis McClure and Robert O. Poytonmitochondrial matrix

Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located both in the cytosol and

published online December 20, 2004J. Biol. Chem.

10.1074/jbc.M411478200Access the most updated version of this article at doi:

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Documents

Yeast Flavohemoglobin , a Nitric Oxide Oxidoreductase, is ... · heme binding domain, the “myoglobin fold” (2). Aside from these common features hemoglobins Aside from these common