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Journal of Bacteriology, August 1998, p. 4051-4055, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Cytosolically Directed NADH Dehydrogenase
in Mitochondria of Saccharomyces cerevisiae
W. Curtis
Small and
Lee
McAlister-Henn*
Department of Biochemistry, University of
Texas Health Science Center, San Antonio, Texas 78284-7760
Received 1 May 1998/Accepted 9 June 1998
 |
ABSTRACT |
The reoxidation of NADH generated in reactions within the
mitochondrial matrix of Saccharomyces cerevisiae is
catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem.
195:857-862, 1991). Gene disruption analysis was used to examine
possible metabolic functions of two proteins encoded by open
reading frames having significant primary sequence similarity to
Ndi1p. Disruption of the gene designated NDH1 results
in a threefold reduction in total mitochondrial NADH dehydrogenase
activity in cells cultivated with glucose and in a fourfold reduction
in the respiration of isolated mitochondria with NADH as the
substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase
capable of using exogenous NADH. Disruption of a closely related gene
designated NDH2 has no effect on these properties. Growth
phenotype analyses suggest that the external NADH dehydrogenase
activity of Ndh1p is important for optimum cellular growth with a
number of nonfermentable carbon sources, including ethanol.
Codisruption of NDH1 and genes encoding malate
dehydrogenases essentially eliminates growth on nonfermentable carbon
sources, suggesting that the external mitochondrial NADH dehydrogenase
and the malate-aspartate shuttle may both contribute to reoxidation of
cytosolic NADH under these growth conditions.
| |
INTRODUCTION |
In eukaryotic cells, reoxidation of
NADH generated by catabolic reactions in the cytosol requires
fermentation reactions or delivery of reducing equivalents to the
mitochondrial electron transport chain. Early studies indicated that
isolated mitochondria from Saccharomyces cerevisiae are
capable of oxidation of exogenous NADH (24, 25), as are
mitochondria from plants and fungi. This suggests the existence of an
externally directed NADH dehydrogenase that may catalyze the metabolic
oxidation of cytosolic NADH. This direct oxidation could occur in lieu
of indirect oxidation via the malate-aspartate shuttle cycle
characteristic of mammalian cells. Additional studies have
characterized at least two separate enzymatic activities in yeast
mitochondria capable of delivery of reducing equivalents from NADH to
the respiratory chain (5, 7, 20). These dehydrogenases
differ from complex I of mammalian mitochondria in several ways,
including the absence of coupling to site I phosphorylation,
insensitivity to rotenone or piericidin, and the absence of an
iron-sulfur redox center.
The best characterized of yeast NADH dehydrogenases is encoded by the
NDI1 gene (genome designation YML120c)
isolated and characterized by de Vries et al. (10). The
purified Ndi1 protein is composed of a single subunit
(Mr, 53,000) and contains noncovalently linked
flavin adenine dinucleotide (8). The protein has an amino-terminal mitochondrial targeting sequence which is removed upon
import (10). Analysis of null mutants containing a
disruption of the NDI1 gene indicated that the enzyme
is primarily involved in oxidation of NADH generated within the
mitochondrial matrix (20). Gene disruption was found to have
no effect on growth with fermentable carbon sources or ethanol but to
reduce or eliminate growth on lactate, pyruvate, or acetate.
In contrast to the internal NADH dehydrogenase represented by Ndi1p,
the identity of the external dehydrogenase(s) has not been established.
An activity oriented toward the intermembrane space has been
described (5, 7, 13), but the protein has not been
isolated. The S. cerevisiae genome sequence
reveals two open reading frames (ORFs), YMR145c and
YDL085w, with significant homology to NDI1.
The current study describes effects of disruption of the corresponding
genes to establish if either or both encode an externally directed NADH
dehydrogenase.
Interest in the external NADH dehydrogenase derives, in part, from
aspects of phenotypes determined for yeast mutants constructed by
disruption of genes for cytosolic and mitochondrial isozymes of malate
dehydrogenase. For example, disruption of the MDH1 gene encoding the mitochondrial tricarboxylic acid cycle enzyme produces an
inability to utilize acetate as a carbon source (21). Since this disruption does not eliminate growth with ethanol, it was speculated that sufficient energy for growth might be provided by
delivery of reducing equivalents from reactions catalyzed by alcohol
and aldehyde dehydrogenases and that this delivery could involve the
external NADH dehydrogenase. Also, codisruption of MDH1 and
MDH2, the gene encoding the cytosolic enzyme, does not significantly affect growth on glucose or eliminate growth with other
nonfermentable carbon sources like ethanol or glycerol-lactate (23), suggesting a metabolic alternative to the
malate-aspartate shuttle. Thus, in addition to examining the
metabolic effects of disruption of genes encoding putative external
NADH dehydrogenases, we have also examined phenotypes resulting from
codisruption of these genes with MDH1 and MDH2.
Our results suggest that shuttle cycle functions and external NADH
oxidation may, in part, be interchangeable.
| |
MATERIALS AND METHODS |
Yeast strains and growth conditions.
The S. cerevisiae strains used in this study were the parental haploid
strain S173-6B (MATa leu2-3,112 his3-1 ura3-52 trp1-289) (2) and derivatives of this strain containing
specific gene disruptions. These include strains containing a deletion and a HIS3 insertion in the chromosomal locus for
MDH1 or MDH2, constructed as previously reported
(21, 23), and strains containing disruptions of the
NDH1 and NDH2 loci, constructed as described below.
Yeast strains were cultivated in rich YP medium (1% yeast extract, 2%
Bacto Peptone) or in minimal YNB medium (0.17% yeast nitrogen base,
0.5% ammonium sulfate, pH 6.0) containing supplements (20 µg/ml) to
satisfy auxotrophic requirements for growth. Carbon sources used at a
concentration of 3% were glucose, ethanol, and glycerol plus lactate.
Potassium acetate (1%) was also used as a carbon source. With
cultivation in liquid medium, 50 mM potassium phosphate (pH 6.8) was
added as a buffer to prevent cell clumping. Culture growth rates
were monitored by turbidity measurements using a Klett photometer
(Manostat Corp., New York, N.Y.) with a KS-59 filter. For
growth curves, cultures were inoculated from fresh logarithmic-phase
starter cultures to an initial density of 40 Klett units.
Disruption of NDH1 and NDH2
loci.
The S. cerevisiae NDH1 gene (ORF
YMR145c) was amplified from a yeast genomic DNA library by
PCR. Oligonucleotides
(5'-GGGGGGATCCATGATTAGACAATCATTAATGAAAACAGTG-3' and
5'-CCCCCGAATTCCTAGATAGATGAATCTCTACCCAAGAAATAAAC-3'), used as
primers, were derived from sequences from the 5' and 3' termini of the
coding region. The resulting PCR product was digested with BamHI and EcoRI and ligated into the yeast
shuttle vector pRS426 (28). The identity of the
subcloned PCR product was confirmed by partial nucleotide sequence
analysis (32). For disruption, the yeast TRP1
gene was subcloned by blunt-end ligation into a unique EheI
restriction site in the NDH1 coding region. The
BamHI/EcoRI DNA fragment containing the
interrupted coding region was used for transformation of strain S173-6B
and one-step gene disruption (27).
The
NDH2 gene (ORF
YDL085w) was disrupted by the
heterologous cassette method of Wach et al. (
33). The
kanMX4 selection
gene was amplified by PCR using
plasmid pFA6-
kanMX4 as the template.
The oligonucleotides
used as primers contained terminal 5' and
3' sequences
derived from the
NDH2 coding region. The PCR product
was used for direct transformation of various yeast strains and
transformants identified as colonies resistant to geneticin (200
µg/ml). Yeast transformations were conducted by using lithium
acetate
(
12,
15). Chromosomal gene disruptions were confirmed
by
Southern blot analysis (
31) of genomic DNA isolated from
Trp
+ or geneticin-resistant colonies.
Northern blot analysis of
NDH1 expression was conducted by
using RNA samples isolated as described in Rose et al. (
26)
from
cultures of the parental strain harvested during logarithmic
growth
on YP medium with various carbon sources. DNA probes were
prepared
by the random primer labeling procedure (
14) by
using the
BamHI/
EcoRI
fragment from the cloned
NDH1 gene and a 1.1-kbp fragment from
the yeast actin gene.
Densitometry was used for quantitative comparison
of autoradiographic
results.
Cellular fractionation and protein analyses.
For
cellular fractionation, 200-ml cultures were grown with YP medium
containing glucose or glycerol-lactate as the carbon source to an
optical density at 600 nm of 1.0 to 2.0. Growth was terminated by
addition of 0.2-mg/ml cycloheximide and incubation on ice for 15 min.
Cell pellets were used for subcellular fractionation as described by
Daum et al. (6), producing an organellar pellet containing mitochondria and peroxisomes and a soluble cytosolic fraction. For enzymatic assays, the organellar pellet was resuspended in 0.5 to 2.0 ml of 10 mM Tris-HCl (pH 7.4)-0.05% (wt/vol)
phenylmethylsulfonyl fluoride and lysed by vortexing with glass beads.
Malate dehydrogenase activity was measured as previously described
(
21). NADH dehydrogenase activity was measured as the
NADH-dependent reduction of cytochrome
c by a modification
of
the method of Sottocasa et al. (
30). Assays were
conducted in
a volume of 1.0 ml containing 50 mM potassium phosphate
(pH 7.4),
2 mM potassium cyanide, and 0.1 mM cytochrome
c.
Assays were initiated
by addition of NADH (or NADPH) to a final
concentration of 0.1
mM and monitored spectrophotometrically at
550 nm. Protein concentrations
were measured by the method of Bradford
(
3) by using bovine
serum albumin as the standard.
Organellar pellets were resuspended for measurements of oxygen
consumption by using a Clark-type oxygen electrode (YSI Inc.,
Yellow
Springs, Ohio) as described by Balcavage et al. (
1).
Respiratory substrates were 5 mM succinate, 5 mM pyruvate plus
5 mM malate, and 0.1 mM NADH (or NADPH). Measurements were made
in the
absence and presence of 0.1 mM ADP, and all respiration
was found to be
sensitive to inhibition by cyanide or azide.
| |
RESULTS |
Disruption of putative NDH genes.
A search of the
S. cerevisiae genome sequence database
revealed two ORFs, YMR145c and YDL085w,
with extensive sequence similarity to the previously identified gene
(NDI1, ORF YML120C) encoding the internal
form of mitochondrial NADH dehydrogenase (20). For
simplicity, the YMR145c and YDL085w ORFs
potentially encoding NADH dehydrogenases are designated
NDH1 and NDH2, respectively. The aligned
primary sequences of Ndh1p and Ndh2p share 62% residue identity and 48 and 46% residue sequence identity, respectively, with Ndi1p. The ORFs
can also encode polypeptides of similar sizes: 513 residues for Ndi1p,
560 for Ndh1p, and 545 for Ndh2p. Among striking differences, as
illustrated in the partial sequence comparison in Fig.
1, are sequences near the amino termini.
Ndh1p and Ndh2p have long amino-terminal extensions with significant
homology beginning at respective residue positions 49 and 39. The amino terminus of Ndi1p is substantially shorter, but the sequence of the
mature polypeptide commences with significant homology to the Ndh1p and
Ndh2p sequences. The 26-residue mitochondrial targeting sequence of
Ndi1p (10) is bracketed. The first 20 to 30 residues of the
amino-terminal extensions of Ndh1p and Ndh2p also contain multiple residues with positively charged and hydroxylated side chains, characteristic of mitochondrial targeting sequences.
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FIG. 1.
Comparison of amino-terminal sequences of yeast NADH
dehydrogenase homologs. Shown is a partial alignment of amino
acid sequences for Ndi1p (20) (ORF YML120c on
chromosome XIII), Ndh1p (ORF YMR145c on chromosome XIII), and Ndh2p
(ORF YDL085w on chromosome IV). Residue identities are indicated by
colons, and similarities are indicated by periods. Brackets enclose the
mitochondrial targeting sequence of Ndi1p (10). The total
number of residues in each ORF is shown in parentheses.
|
|
Gene disruption analysis was conducted to test the enzymatic and
metabolic functions of Ndh1p and Ndh2p. Cassettes for disruption
were
constructed by PCR as described in Materials and Methods.
A haploid
yeast strain containing a disruption of the
NDH1 gene
was
constructed by transformation with a DNA fragment containing
the
NDH1 coding region interrupted by the yeast
TRP1 gene. The
NDH2 genes in this strain and the
parental strain were subsequently
disrupted by replacement with a
kanMX4 gene. Trp
+ and geneticin-resistant
transformants representing single- and
double-mutant strains were
isolated, and the gene disruptions
were verified by Southern blot
analyses.
Initial analyses of activity were conducted by using extracts from
soluble cytosolic and organellar (mitochondrial) fractions
following
cultivation of mutant and parental strains on rich medium
with various
fermentable or nonfermentable carbon sources. Under
all conditions,
NADH dehydrogenase activity was found to be associated
with the
organellar pellet. These assays, which monitor oxidation
of NADH as a
function of cytochrome
c reduction by the
bc1 complex,
were conducted following mechanical
disruption of organellar membranes
and thus are a measure of total
mitochondrial activity. As illustrated
in Table
1, levels of total NADH dehydrogenase
activity associated
with mitochondria from the parental strain are
elevated approximately
threefold with growth on glycerol-lactate
relative to growth on
glucose as the carbon source. Similarly elevated
levels are detected
in mitochondrial fractions from the
NDH mutant strains following
growth on glycerol-lactate,
and differences among the various
strains are not significant. However,
significant differences
in activity are measurable following growth on
glucose. An approximate
threefold decrease in activity is measured for
strains containing
the
NDH1 gene disruption, whereas
disruption of
NDH2 has no measurable
effect on activity
under these conditions. These results are consistent
with previous
reports that Ndi1p activity is derepressed with
growth on
nonfermentable carbon sources (
8). They also suggest
that Ndh1p is the primary contributor to NADH dehydrogenase activity
in
cells grown on glucose. Northern blot analyses (data not shown)
indicate similar levels of
NDH1 mRNA, relative to yeast
actin
mRNA levels, with growth on all of the carbon sources used in
this study, suggesting that expression may be constitutive.
To distinguish relative contributions to oxidation of external NADH,
rates of oxygen consumption with this and other respiratory
substrates
were compared by using mitochondria isolated from cells
grown with
glucose as the carbon source. As shown in Table
2,
rates of oxygen consumption with
succinate are similar for mitochondria
from the parental strain and the
NDH1,
NDH2, and
NDH1
NDH2 disruption strains, whereas rates with pyruvate plus
malate as
the substrate are slightly reduced for strains with the
NDH1 disruption. In contrast, the
NDH1 disruption is associated with
a threefold reduction
in respiratory rates with NADH as the substrate.
For the parental and
NDH2 disruption strains, the approximate
4:1 ratio
of rates with NADH versus succinate is similar to published
values for
mitochondria from glucose-grown cells (
13,
29).
However, this ratio for mitochondria from the
NDH1
and
NDH1 NDH2 disruption strains is reduced
approximately fourfold. Thus,
these data suggest that Ndh1p is the
dehydrogenase primarily responsible
for delivery of reducing
equivalents from external NADH to the
respiratory chain. The residual
oxidative capacity with NADH of
mitochondria isolated from
NDH1 strains may be due to another
external activity;
however, the inherent fragility of isolated
mitochondria could also
account for some access to the internal
NADH dehydrogenase.
There is at least one report that yeast mitochondria may be capable of
oxidation of NADPH (
11). We measured NADPH-dependent
reduction of cytochrome
c by mitochondrial extracts from the
strains
listed in Table
1. Levels of activity in extracts were
approximately
10% of that measured for NADH in the parental extract,
and no
significant differences were detected among various strains.
Also,
we found no measurable oxidation of NADPH by respiring
mitochondria
isolated from any of the strains in this study. Thus, any
mitochondrial
oxidation of NADPH likely occurs internally and may be
attributable
to Ndi1p. This would be analogous to utilization of both
NADH
and NADPH by an internal dehydrogenase in plant mitochondria
(
22).
Growth phenotypes associated with NDH and
MDH gene disruptions.
The effects of NDH1
and NDH2 gene disruptions on growth rates with various
carbon sources were examined by using logarithmically growing cultures
in both rich and minimal media. As shown in Table 3, experiment A, disruption of either or
both genes was found to have no effect on growth rates with glucose.
Also, single disruption of NDH2 does not significantly alter
growth rates relative to the parental strain on any carbon source
tested. However, disruption of NDH1, alone or in combination
with disruption of NDH2, was found to reduce growth rates
with nonfermentable carbon sources, including glycerol-lactate (data
not shown) and ethanol. The most dramatic effect is an ~twofold
lengthening of generation time with ethanol as the carbon source in
minimal medium (Table 3, experiment A); this effect is also observed
but is less pronounced with rich medium. These results are consistent
with an important role for Ndh1p in reoxidation of NADH, which may
accumulate to significant levels in the cytosol during ethanol
utilization due to the reaction catalyzed by cytosolic alcohol
dehydrogenase (34).
That
NDH1 strains grow on nonfermentable carbon
sources, albeit at reduced rates, suggests that reoxidation of
cytosolic
NADH by this enzyme is not essential or that an alternative
mechanism
is available. To test if one alternative could be the
malate-aspartate
shuttle, the
NDH1 gene disruption was
introduced into yeast strains
containing disruptions of the genes
encoding the mitochondrial
(Mdh1p) or cytosolic (Mdh2p)
isozyme of malate dehydrogenase,
an enzymatic participant in the
shuttle cycle. As shown in Table
4,
disruption of
NDH1 in these strains results in an
approximate
threefold reduction in NADH dehydrogenase activity in
mitochondria
from cells grown with glucose analogous to that described
for
the parental strain disruption. Disruption of
MDH1 or
MDH2 reduces
respective compartmental activity by 3- to
10-fold in subcellular
fractions, as previously reported (
21,
23).
As illustrated in Table
3, experiment B, and as previously reported
(
23), the most dramatic phenotype associated with disruption
of
MDH2 is an inability to grow with ethanol as the carbon
source
on minimal medium. Since growth with ethanol on rich medium is
less affected, this phenotype may indicate a function in
gluconeogenesis
for production of C4 metabolites. Codisruption of
MDH2 and
NDH1 was found to essentially eliminate
growth on other nonfermentable
carbon sources with either rich or
minimal medium. One interpretation
of this "synthetic" phenotype is
that Mdh2p and Ndh1p have complementary
functions in reoxidation of
cytosolic NADH during growth on nonfermentable
carbon sources.
The most dramatic phenotype associated with disruption of
MDH1 is inability to grow with acetate as the carbon source
on either
rich or minimal medium (
21), while growth with
other nonfermentable
carbon sources is reduced. The acetate growth
phenotype, also
observed for mutants with a disruption in the
CIT1 gene encoding
mitochondrial citrate synthase
(
17), has been interpreted as
an energetic defect. Since
MDH1 and
CIT1 mutants grow well on
rich medium
with ethanol, production of NADH during ethanol utilization
and
conversion to acetate was proposed to allow bypass of the
energetic
defect. Codisruption of
MDH1 and
NDH1 was found
in this
study (Table
3, experiment C) to significantly reduce growth
with ethanol as the carbon source on both rich and minimal media,
suggesting that Ndh1p is responsible for this bypass, allowing
delivery
of cytosolic reducing equivalents for energy production.
In contrast, codisruption of
NDH2 and
MDH1 or
MDH2 was found to have no effect on growth rates measured
for the malate dehydrogenase
mutants, suggesting no overlap of
the physiological functions
of these enzymes.
| |
DISCUSSION |
The functions of two proteins with extensive primary sequence
homology with yeast Ndi1p, the matrix-oriented mitochondrial NADH
dehydrogenase, were investigated by disruption of the
corresponding genes in S. cerevisiae. Evidence is presented
that the protein designated Ndh1p catalyzes the oxidation of
cytosolic NADH or of exogenous NADH in the case of isolated
mitochondria, but no physiological function has been determined for
Ndh2p. This family of homologous proteins in yeast shares some
similarity with single-subunit NADH dehydrogenase of Escherichia
coli (20), but these proteins appear to be
evolutionarily and structurally distinct from the multisubunit complex
I ubiquinone oxidoreductases characteristic of mammalian cells.
Independent function of yeast Ndi1p has been demonstrated by its
purification as a single polypeptide (8) and by
demonstration of function in the bacterial respiratory chain when
NDI1 is expressed in E. coli (18).
Disruption of the NDH1 gene results in an approximate
threefold reduction in rates of oxygen consumption by isolated
mitochondria with NADH but has little effect on respiration with
pyruvate-malate as the respiratory substrate, suggesting that the
Ndh1p catalytic function primarily involves external reducing
equivalents. Essentially opposite effects were obtained with disruption
of NDI1 (20), i.e., loss of respiration with
pyruvate-malate but not with NADH. Mitochondria from both types of
mutants utilize succinate due to independent delivery of reducing
equivalents from flavin adenine dinucleotide-H2. In terms
of contribution to total mitochondrial activity, disruption of
NDH1 (and NDH2) has little effect on elevated levels in extracts from cells grown with nonfermentable carbon sources.
Differences in NADH dehydrogenase activity attributable to Ndh1p are
measurable only with growth on glucose, a condition associated with
repressed expression of Ndi1p (8).
Despite lower levels of Ndh1p activity relative to Ndi1p with growth on
nonfermentable carbon sources, the phenotypes exhibited by haploid
yeast strains containing a disruption of the NDH1 gene suggest that direct oxidation of cytosolic NADH is crucial for optimum
growth under these conditions. Ndi1p is also important for optimum
growth on most nonfermentable carbon sources (20). In fact,
the major difference between strains with corresponding gene
disruptions appears to be the reduction in growth with ethanol associated with NDH1 disruption; this phenotype and its
absence with NDI1 disruption suggest the importance of the
external activity when net oxidation is required for assimilation of a
carbon source. Growth with glucose as a carbon source is not affected
by disruption of any of the genes encoding putative NADH dehydrogenases
(Table 3) (20). Also, as shown in this study, concomitant
disruption of NDH1 and NDH2 and repressed
expression of Ndi1 do not reduce glucose growth rates, suggesting the
sufficiency of fermentation reactions for reoxidation of NADH.
It has been proposed that the malate-aspartate shuttle cycle may
not be functional in yeast due to the capacity for direct oxidation of
cytosolic NADH (9, 16). We have attempted to address this question by analysis of mutants lacking both Ndh1p and
either the cytosolic or mitochondrial isozyme of malate dehydrogenase. One aspect of growth phenotypes exhibited by strains lacking cytosolic Mdh2p is reduced growth on rich medium with ethanol as the carbon source (Table 3). This is distinct from the auxotrophic
phenotype exhibited on minimal medium. Since codisruption of
MDH2 and NDH1 eliminates the residual
capacity of the single-disruption mutant strains for growth on ethanol
with rich medium, it is possible that the two gene products provide
complementary or additive physiological functions, and it seems
reasonable to assume that the common function is oxidation of cytosolic
NADH. Similarly, disruption of MDH1 only slightly reduces
growth with ethanol on rich or minimal medium, indicating that an
intact tricarboxylic acid cycle is not essential under these
conditions. Codisruption with NDH1, however, eliminates ethanol growth, indicating that redox balance may require either an
intact cycle-shuttle function or the external dehydrogenase activity. Collectively, these results suggest that both direct and
indirect paths for oxidation of NADH may be operative. An alternative
possibility is that the malate dehydrogenases are important substrate
sources for the external NADH dehydrogenase.
A potential alternative for oxidation of cytosolic NADH is the glycerol
phosphate shuttle. However, a recent study by Larsson et al.
(19) indicates that although this shuttle is very active in yeast cells grown with ethanol, disruption of genes encoding enzymatic components of the shuttle has no effect on ethanol growth rates. Thus, this shuttle is not physiologically essential,
at least under aerobic conditions. Of interest for future studies are
correlations between the functions of the different shuttle cycles. Also, although no deleterious consequences for growth have been found to correlate with disruption of the NDH2
gene, further examination of expression and other growth conditions is
required to elucidate or eliminate a possible metabolic function. Along
these lines, it is of interest to determine the identity of a
high-molecular-weight complex with NADH dehydrogenase activity which is
reportedly induced by starvation (4). Finally, the yeast
NADH dehydrogenases provide an interesting system for examination of
mechanisms for differential compartmental-membrane localization.
| |
ACKNOWLEDGMENT |
This work was supported by Public Health Service grants GM33218
and GM51265 from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Texas Health Science Center, San
Antonio, TX 78284-7760. Phone: (210) 567-3782. Fax:
567-6595. E-mail: henn{at}uthscsa.edu.
| |
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Journal of Bacteriology, August 1998, p. 4051-4055, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
