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Originally published In Press as doi:10.1074/jbc.M107016200 on September 26, 2001
J. Biol. Chem., Vol. 276, Issue 48, 45403-45407, November 30, 2001
Heme A Is Not Essential for Assembly of the Subunits of
Cytochrome c Oxidase of Rhodobacter
sphaeroides*
Laree
Hiser and
Jonathan P.
Hosler
From the Department of Biochemistry, The University of
Mississippi Medical Center, Jackson, Mississippi 39216
Received for publication, July 24, 2001, and in revised form, September 24, 2001
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ABSTRACT |
The aa3-type cytochrome
c oxidase of Rhodobacter sphaeroides, a
proteobacterium of the  subgroup, is structurally similar to the
core subunits of the terminal oxidase in the mitochondrial electron
transport chain. Subunit I, the product of the coxI gene, normally binds two heme A molecules. A deletion of cox10,
the gene for the farnesyltransferase required for heme A synthesis, did
not prevent high level accumulation of subunit I in the cytoplasmic membrane. Thus, subunit I can be expressed and stably inserted into the
cytoplasmic membrane in the absence of heme A. Aposubunit I was
purified via affinity chromatography to a polyhistidine tag. Copurification of subunits II and III with aposubunit I indicated that assembly of the core oxidase complex occurred without the binding
of heme A. In addition to formation of the apooxidase containing all
three large structural proteins, CoxI-II and CoxI-III heterodimers were
isolated from cox10 deletion strains harboring expression
plasmids with coxI and coxII or with
coxI and coxIII, respectively. This
demonstrated that subunit assembly of the apoenzyme was not an
inherently ordered or sequential process. Thus, multiple paths must be
considered for understanding the assembly of this integral membrane
metalloprotein complex.
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INTRODUCTION |
Cytochrome c oxidase (complex IV), the terminal member
of the electron transport chain in mitochondria, is essential for
respiration. A closely related aa3-type oxidase
is found in members of the subgroup of the proteobacteria,
including Rhodobacter sphaeroides. Defective assembly of the
bacterial aa3-type oxidase does not inhibit
growth because of the presence of alternative terminal oxidases in the
cell (1-3). Thus, assembly of cytochrome c oxidase can be
studied in vivo in R. sphaeroides. A variety of
human diseases, including myopathies and neuropathies, are associated
with oxidase deficiencies that result from a failure of the
multisubunit oxidase to properly assemble (reviewed in Ref. 4).
The subunit composition of the aa3-type oxidase
of R. sphaeroides is ideal for studying assembly of the
catalytic core complex. The bacterial oxidase is composed of only four
subunits (5), whereas the mitochondrial oxidase has up to 13 different
polypeptides encoded by both nuclear and mitochondrial genes (6). The
three largest subunits of the bacterial oxidase are integral membrane proteins homologous to the subunits synthesized in mitochondria (7-9).
Both prokaryotic and eukaryotic oxidases contain tightly bound
cofactors that are necessary for electron transport (10, 11). Two
molecules of heme A, six-coordinate heme a and
five-coordinate heme a3, and a copper atom
(CuB) are in subunit I; two additional copper atoms form
the CuA site in subunit II (5, 12-14).
Heme A is derived from heme B (iron protoporphyrin IX) with heme O as a
probable intermediate (15). Conversion of the vinyl group at position 2 of the heme B porphyrin ring to a hydroxyfarnesyl moiety by the
farnesyltransferase, Cox10p, forms heme O (16, 17). Hemes A and O both
contain the hydrophobic farnesyl group but are readily distinguished by
optical spectroscopy because heme A has a formyl rather than a methyl
group at position 8 of the porphyrin ring.
Many soluble hemoproteins require heme to achieve a native fold but
assume a partially folded state in the absence of heme (Ref. 18 and
references therein). The role that heme A plays in the folding and
assembly of the integral membrane aa3-type oxidase complex is not well defined. An early investigation using fetal
rat liver concludes that apocytochrome c oxidase fails to accumulate in the absence of  -aminolevulinic acid, a precursor of
all hemes (19). Similar observations have been made in Paracoccus denitrificans (20) and Saccharomyces cerevisiae (21)
cox10 mutants. In the yeast mutants, subunit I is
efficiently translated but is nearly absent in steady-state
mitochondria (21). These studies suggest that heme A is necessary for
insertion and/or stability of subunit I in mitochondrial membranes. In
fact, significant amounts of free subunit I are observed in bacterial
membranes when heme A is present (22, 23). Subunit I is relatively
abundant (72% of control) in mitochondria of patients with a
cox10 mutation encoding a partially active protein (24).
Taken together, these studies raise the question of what role heme A
plays in the assembly of cytochrome c oxidase.
In this report, the ability of subunit I to accumulate in the R. sphaeroides membrane in the absence of heme A was studied. An
affinity tag on subunit I was used to isolate partially assembled forms
of cytochrome c oxidase from cells lacking the genes for the
assembly factors Cox10p and Cox11p (8). Heme A was absent because of
the cox10 deletion; Cox11p is necessary for the formation of
CuB (25). The ability of subunit I lacking heme A to
associate with the other members of the protein complex was
investigated by expressing subunit I with subunits II and/or III.
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EXPERIMENTAL PROCEDURES |
Plasmids and Strains--
Plasmids with various combinations of
the genes for R. sphaeroides cytochrome c oxidase
subunits I, II, and III (see Table I) were constructed using standard
molecular techniques (26). Plasmid pYJ123H contains the
coxI-His gene and the coxII/III operon (coxII, cox10, cox11, and
coxIII) in pUC19 (27). Deletions of 4.3- or
3.3-kb1 SmaI
fragments from pYJ123H were made, respectively, to create plasmids
containing only coxI-His (pJH310) or coxI-His and
coxIII both driven off the coxI promoter
(pJH103H). For expression in R. sphaeroides strain YZ200
(27), the low copy, broad host range vector pRK415-1 (28) was used. The
2.5-kb EcoRI-HindIII fragment from pJH310 was
inserted into the multiple cloning site of pRK415-1 to create pWA302.
Similarly, the 3.4-kb EcoRI-HindIII fragment of
pJH103H was placed into pRK415-1 to make pAH103H.
Plasmid pMB307 is related to pYJ123H but
does not contain coxIII (23). Site-directed mutagenesis was
used to change two nucleotides to introduce a SmaI site 20 nucleotides 3' of the cox10 initiation codon. The resulting
plasmid was named pMB307a. Deletion of the 1.6-kb SmaI
fragment from pMB307a left coxI-His and coxII on
opposite strands, each under the control of its natural promoter. This
plasmid was designated pWA300. The addition of a 956-base pair
SmaI fragment from pMB301 (see below) to pWA300 created
pAH123 10,11, which contains coxI-His on one DNA strand and coxII and coxIII behind the coxII
promoter on the opposite strand. The plasmid pMB301 was created by
ligating the 956-base pair SmaI fragment from pYJ100 (27)
into the multiple cloning site of pBC SK + (Stratagene). The expression
vectors pRKpWA300 and pRKpAH12310,11 were derived from pWA300 and
pAH12310,11, respectively, by placing 3.7- or 4.6-kb
EcoRI-HindIII fragments into pRK415-1. To create
the control plasmid containing only coxII and
coxIII, a 1.15-kb SalI fragment internal to
coxI-His was deleted from pAH12310,11 removing greater
than 60% of the coxI open reading frame and creating
pLH339. The 3.5-kb EcoRI-HindIII fragment of pLH339 was then placed into pRK415-1 to form pLH347. Each of the pRK415-based plasmids was conjugated into R. sphaeroides
strain YZ200 by the published method (29).
Bacterial Growth and Oxidase Purification--
R.
sphaeroides cells were grown in minimal medium to the late
exponential phase (25). Protein was purified by affinity chromatography on Ni-NTA-agarose from cytoplasmic membranes solubilized in
N-dodecyl- -D-maltoside as previously
described (25) except that the concentration of KCl in the purification
buffers was 150 mM rather than 40 mM KCl. This
change in the salt concentration did not significantly affect the
ability to purify the oxidase subcomplexes.
Electrophoresis and Immunoblots--
Electrophoresis was
conducted in 14% polyacrylamide gels containing sodium dodecyl sulfate
and 6 M urea (30). The proteins were visualized by rapid
staining with Coomassie Blue (31). For immunoblots, the proteins were
transferred to nitrocellulose and probed essentially as described (32).
For detection of subunit I, a 1:5000 dilution of a monoclonal antibody
against polyhistidine (Sigma) was used with colorimetric detection. For
detection of subunit II, a polyclonal primary antibody raised against
subunit II of the R. sphaeroides aa3-type
oxidase2 was used at a 1:400
dilution with protein A-125I as the secondary reagent.
Cell Labeling--
R. sphaeroides cells were grown in
minimal medium to the exponential phase (OD660 = ~0.3).
The cells were then washed with minimal medium prepared with chloride
rather than sulfate salts and incubated in this medium for 4 h to
decrease the pool of free methionine and cysteine. Radiolabeled
methionine and cysteine were then added to the culture (1 mCi/50 ml) in
the form of EasyTag EXPRESS (PerkinElmer Life Sciences). Growth was
rapidly stopped after 10 min by the addition of 0.01% chloramphenicol,
2% NaN3, 15 mM EDTA and placing the cells on
ice. The culture was then divided into six equal portions for
processing. After washing the cells, the extracts were prepared by
vortexing in the presence of glass beads and the protease inhibitor,
phenylmethylsulfonyl fluoride. Membranes were isolated by
centrifugation at 350,000 × g for 20 min. Washed
membranes were solubilized in
N-dodecyl--D-maltoside, and the
polyhistidine-tagged subunit I was bound to and eluted from
Ni-NTA-agarose in a batch process in a microcentrifuge tube using a
procedure similar to that used for column purification. The entire
eluted fraction was prepared for denaturing gel electrophoresis, and
fixed gels were exposed to a phosphor screen and analyzed using a Storm
860 PhosphorImager and ImageQuant software (Molecular Dynamics).
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RESULTS |
Isolation of Subunit I Lacking Heme A--
The necessity of heme A
for steady-state accumulation of subunit I in the cytoplasmic membrane
of R. sphaeroides was investigated using a strain (YZ200)
with a genomic deletion of the coxII/III operon containing
the coxII, cox10, cox11, and
coxIII genes (27). Because the product of the
cox10 gene, Cox10p, is required for heme A synthesis (33),
YZ200 membranes had only b- and c-type cytochromes (Fig. 1). A low copy, broad
host range expression vector containing the gene for subunit I with a
carboxyl-terminal His6 tag was used to express subunit I in
YZ200 in a form that could be readily purified using Ni-NTA affinity
chromatography. As expected, the cytochrome a peak (~606
nm) was absent from the reduced minus oxidized membrane spectrum (Fig.
1, CoxI) because of the absence of Cox10p. However, a
polypeptide was isolated that migrated on a denaturing polyacrylamide
gel like wild-type subunit I and was identified as the product of the
coxI-His gene using an antibody against the His6
tag (Fig. 2, lane 2).
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Fig. 1.
Absence of the
aa3-type oxidase in cox10
deletion mutants. Dithionite reduced minus ferricyanide
oxidized spectra of membranes of R. sphaeroides strain YZ200
harboring no plasmid (YZ200), pRK415-1 (Vector), pWA302 (CoxI), or
pRKpAH1H32 (WT) were recorded at room temperature using a Hitachi
U-3000 UV-visible spectrophotometer. The spectra were normalized to the
magnitude of the cytochrome b peak. The peaks labeled
a, b, and c are attributed to
a-, b-, and c-type cytochromes,
respectively.
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Fig. 2.
Isolated oxidase subunits. The proteins
were isolated from approximately equal numbers of R. sphaeroides strain YZ200 cells expressing various plasmids listed
in Table I, the vector pRK415-1 (column 1), or pRKpAH1H32
(WT; 25). Four separate gels were used for electrophoresis with loading
of samples normalized to the percentage of material eluted from the
affinity column. Less of the normal oxidase (WT) was used to give
approximately equal signal intensities. Following electrophoresis,
detection of the oxidase subunits was by immunoblotting (CoxIp and
CoxIIp) or by staining with Coomassie (CoxIIIp and lanes
7 and 8).
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The ability to purify the subunit I polypeptide from a strain with a
cox10 deletion demonstrated that the expression and membrane insertion of this protein was independent of heme A production. The
possibility that another type of heme was substituted for heme A was
addressed by measuring the heme content of the isolated protein using
the pyridine hemochrome method (34). Heme B was identified in minor
molar amounts (about 5% that of total protein) in protein isolated
from the strain expressing CoxI. However, a similar amount of heme B
was also found in a control experiment using membranes from strain
YZ200 harboring only the parent vector (pRK415-1). Thus, heme B was
likely to have been present as a minor cytochrome b
contaminant that bound nonspecifically to the metal chelating resin.
The other prosthetic group of subunit I, CuB, was likely to
be absent because of the deletion of the cox11 gene (25).
Thus, the isolated product was presumed to be aposubunit I,
i.e. lacking both hemes and CuB. Throughout this
report, the term "aposubunit I" will refer to the product of the
coxI gene expressed in cells lacking cox10 and
cox11.
Association of Aposubunit I with Subunits II and III--
The
ability of R. sphaeroides aposubunit I to associate with
subunits II and III was ascertained using a copurification assay. Membrane proteins from cells expressing plasmid-borne
coxI-His, coxII, and coxIII genes
(CoxI-II-III; Table I) were subjected to
affinity chromatography on Ni-NTA-agarose. The eluted material contained all three subunits, which were identified by gel
electrophoresis and immunoblotting (Fig. 2, lanes 5 and
8). Because only aposubunit I contained the His6
tag, the isolation of subunits II and III via histidine affinity
chromatography indicated formation of a complex containing all three
subunits. Subunits II and III were not retained on Ni-NTA-agarose when
they were expressed from CoxII/III (Table I) in the absence of
polyhistidine-tagged subunit I (data not shown). Thus, the
copurification of subunits II and III with aposubunit I demonstrated
that formation of the three-subunit oxidase complex was independent of
heme A insertion into subunit I. This CoxI-II-III complex was termed
"apooxidase" because, in addition to the lack of heme A, metal
analysis showed substoichiometric amounts of copper (data not shown).
This suggested that there was little or no CuA in subunit
II. CuB was presumed to be absent because of the
cox11 deletion (25).
Expression of Aposubunit I and Extent of Complex
Formation--
The isolation of the apooxidase was a novel result, but
the significance of this observation to the normal assembly of
cytochrome c oxidase remains to be determined. To begin to
address this question, the extent of complex formation was compared in
strains that differed only by the presence or absence of
cox10 and cox11 genes on the expression plasmid.
The cells were labeled in vivo with
35S-containing amino acids. Complexes containing the
product of the coxI-His gene were isolated from cell
membranes using Ni-NTA-agarose. Expression of the subunit I polypeptide
and the extent of complex formation were evaluated after 10 min of cell
labeling (Fig. 3). Aposubunit I was
expressed to a level at least equal to that of subunit I in the
wild-type strain (Fig. 3, A and B). All three subunits were detected from both the control strain (WT) and the strain
lacking heme A (Fig. 3A, CoxI-II-III). Because of
the low subunit II signal, attributed to a relatively small number of methionines and cysteines, subunit III was chosen for the purpose of
quantitation. Approximately 30% of the newly translated subunit I from
cells containing heme A (WT) and 10% of the aposubunit I from cells
lacking heme A (CoxI-II-III) had associated with labeled subunit III
(Fig. 3C). Because of the possible existence of an unlabeled
pool of subunit III available for assembly, these numbers were taken to
be a conservative estimate of the extent of complex formation. One
possible explanation for the observed difference between formation of
the apooxidase and the holooxidase control is a decreased affinity of
subunit III for subunit I in the absence of heme A.
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Fig. 3.
Extent of complex formation. Cell
cultures of R. sphaeroides strain YZ200 cells containing
plasmid pRKpAH1H32 (WT) or pRKpAH12310,11 (CoxI-II-III) were labeled
with L-[35S]methionine and
L-[35S]cysteine in vivo for 10 min. Complexes containing the polyhistidine-tagged subunit I (CoxIp)
were isolated using a metal chelating resin. A, denaturing
SDS-polyacrylamide gel electrophoresis was used to separate the
isolated complexes into their component subunits (CoxIp, CoxIIp,
and CoxIIIp). Subunit II (CoxIIp) was found in multiple bands because
of posttranslational processing events (27). The image shown is from a
PhosphorImager. B, the amount of subunit I from six
replicate samples of each strain was determined (mean ± S.D.).
Band intensities were normalized to cell number based on optical
density of the cell cultures. C, the relative amount of
labeled subunit III isolated was calculated using the same samples as
for panel B. Subunit III band intensities were multiplied by
2.46 to account for the number of methionines and cysteines: 32 in
subunit I and 13 in subunit III. Using these normalized values, the
amount of subunit III as a percentage of the amount of
subunit I in the same lane was determined (mean ± S.D.,
n = 6).
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The accumulation of the normal oxidase (WT) and apooxidase
(CoxI-II-III) to comparable levels in the bacterial membrane was indicated by the ability to isolate similar amounts of these proteins from steady-state cell cultures (data not shown). Generally, however, the yield of apooxidase was about 20% of the yield of normal oxidase from the same number of cells. These low recoveries may have resulted from an instability of the apooxidase in vitro; aposubunit I
showed a tendency to precipitate. The smaller scale and more rapid
processing of the pulse-labeled cultures may have minimized the effects
of any such instability. Because a complex of aposubunit I with
subunits II and III formed to a level of at least 30% relative to
formation of a similar complex containing heme A, the role of
aposubunit I in the assembly of the normal oxidase deserves consideration.
Assembly Order--
The assembly pathway for mitochondrial
cytochrome c oxidase is believed to be sequential or ordered
(35, 36). To directly determine the minimal requirements for the
association of subunit I with the other core subunits, copurification
of subunit II or subunit III with aposubunit I was tested. R. sphaeroides cells expressing coxI-His and
coxII or coxI-His and coxIII were
studied. Both CoxI-II and CoxI-III heterodimers were isolated from
cells lacking heme A (Fig. 2, lanes 3 and 4).
This result demonstrated that there was no obligate order of assembly
for the protein subunits because both subunits II and III were
competent to associate with subunit I in the absence of heme A.
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DISCUSSION |
In this study, assembly of the three catalytic core proteins of
cytochrome c oxidase was investigated using the
aa3-type oxidase from R. sphaeroides.
Both aposubunit I and a three-subunit apooxidase were isolated from
strains lacking heme A. These proteins inserted into the cytoplasmic
membrane and accumulated to significant levels (at least 30%) compared
with their heme A-containing counterparts. Thus, the insertion of heme
A was not a prerequisite for subunit assembly of the oxidase.
Conversely, subunit association is not required for insertion of one
molecule of heme A (23). This raises the possibility that heme A
insertion may be able to occur at multiple steps in the assembly process.
A dependence on order for the association of aposubunit I with the
other structural subunits was investigated using strains expressing two
of the subunits in the absence of heme A. Two distinct subcomplexes
containing aposubunit I (CoxI-II and CoxI-III) were isolated (Fig. 2,
lanes 3 and 4). Because aposubunit I could
associate with either subunit II or subunit III in genetically
manipulated strains, it was concluded that there was no obligate order
of subunit assembly. Thus, the possibility of multiple paths must be
considered in determining the assembly process of cytochrome c oxidase in wild-type cells. Insertion of the prosthetic
groups also appears to be unordered because heme A is incorporated into subunit I in the absence of other subunits (23) but is not required for
subunit assembly of several apooxidase forms (i.e. CoxI-II, CoxI-III, and CoxI-II-III).
A "state diagram" is a way of illustrating a finite set of possible
assembly paths where some states (subcomplexes) lie on multiple paths.
There are 32 theoretical oxidase assembly intermediates that contain
subunit I, considering only the three metal centers within subunit I
and the three large structural subunits. These possible partially
assembled oxidase forms are arranged in Fig. 4 based on the number of prosthetic
groups in subunit I and the number of protein subunits such that
assembly proceeds from left to right and components are added one at a
time.
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Fig. 4.
Theoretical oxidase subcomplexes.
Possible assembly intermediates or "states" of oxidase (see text)
are shown. Subunits are designated I, II, and III with the
metal-containing cofactors of subunit I shown as subscripts. Oxidase
forms that have been isolated in this or previous studies (23, 25) are
shown as schematics with labels in bold type.
Subunits I, II, and III are represented by rectangles with
no shading, shading, and
cross-hatching, respectively. The bars indicate
the two hemes, and the circles represent CuB.
The CuA site of subunit II has been omitted for simplicity.
Arrows illustrate the most direct paths connecting
characterized subcomplexes which differ by a single component.
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From this study and others (23, 25), a few of these oxidase forms
(shown as schematics) have been isolated from steady-state membranes,
and the logical paths between them can be inferred. Assuming that none
of these purified subcomplexes lie on dead-end paths, the conclusion
must be made that assembly of the holoenzyme is not a strictly ordered
process. For example, the addition of subunit III can be the initial
step (to form CoxI-III) or the final step between
Iaa3CuB-II
and the normal oxidase,
Iaa3CuB-II-III.
Note that some subcomplexes (e.g. I-II-III) lie on multiple
putative paths. The existence of the majority of the theoretical states is currently undetermined, and the extent to which any given subcomplex or putative path exists in wild-type bacteria remains to be
investigated. Therefore, the partially assembled forms of the oxidase
that have been isolated from genetically manipulated bacteria may or
may not be bona fide assembly intermediates.
The deletion of cox10 and cox11 in R. sphaeroides did not prevent the accumulation of aposubunit I in
bacterial cytoplasmic membranes or its association with subunits II or
III (Fig. 2). Consistent with these results, subunit I with no heme A
accumulates to a low level in mitochondria of a yeast strain with a
disruption of the gene for Cox10p (21). This suggests that prokaryotes and eukaryotes have similar mechanisms for assembly of the cytochrome c oxidase core subunits. Furthermore, the gene for Cox11p
was unaltered in the yeast strain (21), indicating that the genetic alteration critical for the observed phenotypes in the present study
was the cox10 deletion. This is consistent with our
observation that an apooxidase (CoxI-II-III) was isolated from a
R. sphaeroides strain containing the wild-type
cox11 gene and a deletion of cox10 (data not
shown). The posttranslational loss of significant amounts of subunit I
in yeast (21) could be due to in vivo degradation or, as
indicated in the present study, could be explained by an in
vitro instability during sample preparation or by structural alterations that reduce the ability of the epitope to be recognized by
the antiserum. These explanations are also relevant to the report of a
P. denitrificans strain with a deletion of the genes corresponding to coxII, cox10, and
cox11 that does not express immunologically detectable
subunit I (20). It is likely that modest overexpression of the gene for
subunit I and isolation of the protein, as opposed to analysis in the
membrane, significantly enhanced our ability to detect aposubunit I in
this study.
In conclusion, heme A is not essential for the expression, membrane
insertion, or association of the core complex proteins of cytochrome
c oxidase in R. sphaeroides. Furthermore, the
assembly of this heterooligomeric integral membrane complex need not
follow a linear or ordered pathway. The structural similarities between the bacterial and mitochondrial oxidases suggest that these findings may extend to assembly of the human oxidase. A "state model" has been proposed as a paradigm for describing the multiple possible assembly paths.
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ACKNOWLEDGEMENTS |
We are grateful to Dr. Melyssa Bratton,
Alicia Hamer, and Wendy McLean for assistance with plasmid
constructions and to Jimmy Gray and Nathan Nix for cell growth and
membrane purifications. We also thank Dr. Victor Davidson for
critically reading the manuscript and Dr. Charles Woodley for
assistance with in vivo labeling experiments.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM56824 (to J. P. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
601-984-1861; Fax: 601-984-1501; E-mail:
jhosler@biochem.umsmed.edu.
Published, JBC Papers in Press, September 26, 2001, DOI 10.1074/jbc.M107016200
2
J. P. Hosler and S. Ferguson-Miller,
unpublished data.
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ABBREVIATIONS |
The abbreviations used are:
kb, kilobase pairs;
Ni-NTA, nickel-nitrilotriacetic acid;
WT, wild type.
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