1 Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of
Health, Bethesda, MD, 20892, USA
2 Department of Metabolic Diseases, Hoffman-La Roche Inc., 340 Kingsland Street,
Nutley, NJ, 07110, USA
* Present address: Department of General Surgery, Carolinas Medical Center,
Charlotte, NC 28232-2861, USA
Author for correspondence (e-mail:
jah{at}helix.nih.gov)
Accepted 31 October 2002
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Summary |
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Key words: OGT, O-GlcNAc, Glycan-dependent signaling, Mitochondria
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Introduction |
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OGT is encoded by a single gene on the X chromosome
(Akimoto et al., 1999;
Kreppel et al., 1997
;
Lubas and Hanover, 2000
;
Lubas et al., 1997
;
Shafi et al., 2000
). Gene
knock-out experiments have shown that OGT is essential for stem cell viability
(Hanover et al., 2002
) and
embryonic development (Shafi et al.,
2000
). OGT can be divided into three functional domains: an
N-terminal tetratricopeptide region (TPR), a linker and a catalytic
C-terminus. The reported OGT isoforms contain between 9-12 TPRs
(Kreppel et al., 1997
;
Lubas and Hanover, 2000
;
Lubas et al., 1997
). This
conserved TPR domain is present in many proteins and is important for
protein-protein interactions (Blatch and
Lassle, 1999
). Varying the number of TPR domains in OGT affects
its substrate recognition (Lubas and
Hanover, 2000
). The linker region of OGT contains a nuclear
localization sequence, consistent with a nuclear localization
(Kreppel et al., 1997
;
Lubas et al., 1997
). The
proposed catalytic domain of OGT contains conserved amino acid domains that
are present in other glycosyl transferases
(Roos and Hanover, 2000
;
Wrabl and Grishin, 2001
).
Modest deletions in the C-terminus of OGT lead to dramatic reductions in
enzymatic activity, supporting its role in catalysis
(Lubas and Hanover, 2000
).
The terminal product in the hexosamine biosynthetic pathway, UDP-GlcNAc, is
utilized by OGT as a donor. A link between the hexosamine biosynthetic pathway
and cellular glucose sensing by insulin has been established
(Marshall et al., 1991;
Traxinger and Marshall, 1991
;
Traxinger and Marshall, 1992
).
Further, overexpression of either the rate-limiting enzyme of the hexosamine
biosynthetic pathway or OGT in the striated muscle and fat of transgenic mice
leads to insulin resistance (Hebert et
al., 1996
; McClain et al.,
2002
; Tang et al.,
2000
; Veerababu et al.,
2000
). This insulin resistance was phenotypically similar to that
observed in human type 2 diabetes. Additionally, recent studies have
demonstrated a relationship between mitochondrial function, O-GlcNAc
metabolism and maintenance of the diabetic state
(Du et al., 2000
;
Nishikawa et al., 2000
).
Therefore it is likely that OGT, by utilizing the terminal product of
hexosamine biosynthesis, may mediate many of the effects attributed to the
dysregulation of this pathway.
Here we report the existence of two OGT isoforms differing in their subcellular localization. The shorter form associates with mitochondria in both primary and immortilized cells, whereas the longer form is nucleocytoplasmic. The N-terminus of the short form contains mitochondrial targeting information and is essential for mitochondrial localization. These data are discussed in terms of the role of differential targeting of OGT to the mitochondrion and nucleus in the regulation of a hexosamine-dependent signaling pathway.
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Materials and Methods |
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Transfections
HeLa cells were seeded on glass coverslips in six-well plates (Corning
Incorporated, Corning, NY) at 1x105 cells per well. Once
cells were attached to the coverslips, they were transfected with indicated
plasmids, using Fugene 6 (Roche, Indianapolis, IN) according to the
manufacturer's instructions.
Antibodies
Mouse anti-Cytochrome clone 26E3, used at 2 µg/ml, and clone 7H8.2C12,
used at 0.5 µg/ml, were obtained from Zymed Laboratories (South San
Francisco, CA). Anti-myc clone 9E10 (Roche) was used at 2 µg/ml.
Anti-mitochondrial heat shock protein 70, clone JG1 (Affinitiy Bioreagents,
Golden, CO) was used at a dilution of 1:500. Anti-O-linked
N-Acetlyglucosamine, clone RL2 (Affinity Bioreagents) was used at a dilution
of 1:100, and CTD110.6 (Covance, Berkeley CA) was used at 1:1000. Rabbit
polyclonal peptide antiserum was raised against residues 581-600 of mOGT.
Affinity-purified antibodies to OGT were generated and purified according to
published methods (Baskin et al.,
1999; Osborne et al.,
1995
).
Immunolocalization
HeLa cells CCL2 (ATCC, Manassas, VA) were grown as described
(Love et al., 1998). Human
aortic endothelial cells (Cascade Biologics, Portland, OR) were grown and
maintained according to the supplier's instructions. Cells were grown
overnight on glass coverslips, then processed for immunolocalization. All
cells were fixed with 4% formaldehyde (Ladd Research Industries, Williston,
VT) in PBS (Mediatech, Herndon, VA) for 30 minutes at room temperature, washed
three times in PBS, then permeabilized with 0.1% tritonX-100
(Calbiochem-Novabiochem Corporation, La Jolla, CA) for 3 minutes. After
permeabilization, cells were washed three times in PBS and incubated with the
primary antibodies for 1 hour at room temperature with gentle shaking. Primary
antibodies were diluted in 5% goat serum, 2% BSA, in PBS. Following primary
antibody incubation, cells were washed once for 15 minutes, then twice for 5
minutes before addition of appropriate, fluorescently conjugated, secondary
antibodies (JacksonImmuno Research, West Grove, PA) for 1 hour at room
temperature. Where indicated, MitoTracker® Red CM-H2Xros
(Molecular Probes, Eugene, OR) was used to label respiring mitochondria
according to manufacturer's instructions. Cells were then washed three times
in PBS and mounted on glass coverslips using Vectashield antifade mounting
medium (Vector Laboratories, Inc., Burlingame, CA). All images were obtained
using an Axiovert 200M (Carl Zeiss Inc., Thornwood, NY) with an Ultra View
(Perkin Elmer, Wellesley, MA) spinning disk confocal scan head. Images were
captured with a Quantix back-thinned EE57 CCD camera (Roper Scientific,
Trenton, NJ) with binning set at two. All images were processed using OpenLab
software (Improvision, Lexington, MA).
Electron microscopy
Purified mitochondria (4C Biotech, Seneffe, Belgium) were prepared and
imaged by Paragon Biotech, Inc. (Baltimore, MD). Briefly, mitochondrial
pellets were fixed with either 4% paraformaldehyde or 4% paraformaldehyde with
1% glutaraldehyde in 0.1 M phosphate buffer and processed for routine TEM
embedding and sectioning and mounted onto nickel grids. The grids were
incubated in 3% H2O2 in 0.1M PB for 5-15 minutes. After
several rinses in PBS, grids were blocked with 0.5% BSA, 2% normal goat serum
in PBS for 30 minutes. Sections were incubated with rabbit polyclonal OGT
antiserum diluted 1:50 in PBS for 1 hour. Sections were then incubated with
biotinylated, goat anti-rabbit secondary antibody at 1:400 (Vector) in PBS for
30 minutes at room temperature, followed by incubation with strepavidin gold
conjugate (1:20, 10 nm gold particles) for 1 hour and rinsed in distilled
water. The grids were double stained with uranyl acetate and lead citrate and
observed and photographed using a Zeiss electron microscope.
Biochemical isolation and separation of HeLa cells
HeLa cell subcellular fractions were obtained from 4C Biotech. Mitochondria
were separated into soluble proteins and membrane-associated proteins.
Peripheral proteins were removed by incubating mitochondria in 1 M KC1 for 30
minutes on ice, followed by centrifugation at 18,000 g for 30 minutes
at 4°C. The mitochondrial pellet was resuspended at 30 mg/ml in 0.6 M
sorbitol, 20 mM HEPES, pH 7.4, 2 mM MgCl2 supplemented with
Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche). The
mitochondrial suspension was sonicated for three 10 second pulses using a
Ultrasonic Processor XL (Misonix, Inc., Farmingdale, NY). Broken mitochondria
were then centrifuged for 30 minutes at 100,000 g at 4°C to
separate membrane-associated and soluble proteins. The membrane-associated
proteins were then solubilized in 0.2% Triton X-100. The soluble proteins
contained within the supernatent were precipitated by adding 0.0125% (w/v)
sodium deoxycholate, then one fifth of the volume of 72% TCA for 30 minutes
and centrifuged for 30 minutes at 18,000 g. Pellets were washed with
ice cold acetone and recentrifuged. All pellets were resuspended in NuPAGE LDS
sample buffer (Invitrogen). Mitoplasts were generated as described
(Noma et al., 2001). Briefly,
mitochondria were suspended in 20 mM HEPES-KOH, pH 7.4, and then placed in a
test tube on ice for 30 minutes. The mitoplasts were recovered by
centrifugation at 4000 g and then resuspended in 10 mM HEPES-KOH, pH
7.4, containing 220 mM mannnitol and 70 mM sucrose. Alkaline extraction with
carbonate buffer was performed as described previously
(Lynn et al., 2001
). Briefly,
mitochondria and mitoplasts were resuspended in 100 mM sodium carbonate pH 11
for 30 minutes at 4°C, then centrifuged at 100,000 g for 30
minutes. All protein determinations were performed by the BCA assay (Pierce
Biotechnology, Rockford, IL).
Western blot analysis
Protein samples were separated using NuPage® Bis-Tris gels (Invitrogen)
and transferred to a nitrocellulose membrane for immunoblot analysis. Briefly,
nitrocellulose membranes were blocked for 1 hour at room temperature with 5%
nonfat dry milk with 0.1% Tween-20 (Sigma Chemical Corporation, St. Louis,
MO), in Tris-buffered saline (Quality Biological, Incorporated, Gaithersburg,
MD) (TBS-T). Primary antibodies were diluted in TBS-T containing 2% nonfat dry
milk and incubated with the membrane. The horseradish-peroxidase-conjugated
secondary antibodies (Jackson Immuno Research) were diluted in 2% BSA, 0.1%
Tween 20, PBS. All antibody incubations were conducted for 1 hour at room
temperature. Washes were conducted to remove the unbound primary and secondary
antibodies and consisted of two quick washes in TBS-T or PBS-T, then one 15
minute and two 5 minute washes. Membranes were visualized using the
Renaissance Chemiluminescence Reagent Plus Western Blot kit (NENTM Life
Science Products, Inc., Boston, MA) according to the manufacturer's
instructions.
Proteomics tools
The mitochondrial targeting sequence was predicted using iPSORT
(http://www.hypothesiscreator.net/iPSORT/).
The membrane-spanning region was predicted using TopPred 2
(http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html).
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Results |
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|
|
Targeting information in OGT isoforms
In contrast to the expected nuclear localization of OGT, the striking
mitochondrial localization we observed was surprising. The findings further
suggested that the 103 kDa mitochondrial form of the enzyme might represent a
distinct biochemical species (Fig.
1B). Several isoforms of OGT have been described which seem to
arise by alternative splicing of the mammalian ogt gene
(Kreppel et al., 1997;
Lubas et al., 1997
;
Nolte and Muller, 2002
;
Hanover et al., 2002
). These
known isoforms include species with predicted molecular weights of 116
(Kreppel et al., 1997
) and 103
kDa (Lubas et al., 1997
). We
utilized several web-based search tools to identify regions of OGT that might
target either of these isoforms to mitochondria. iPSORT identified a 20
amino-acid stretch at the unique N-terminal end of the 103 kDa isoform, which
we designate mOGT (mitochondrial OGT)
(Lubas et al., 1997
), as the
mitochondrial targeting sequence (Fig.
2A, bottom row). Interestingly, this mitochondrial targeting
sequence was detected only when mOGT was designated as a plant protein,
suggesting that this mitochondrial targeting sequence is somewhat unusual. The
longer isoform of OGT, which we designate ncOGT (nucleocytoplasmic OGT)
(Kreppel et al., 1997
), did
not contain a mitochondrial targeting sequence; instead, ncOGT contains a
unique N-terminus and three additional TPRs when compared to mOGT
(Fig. 2A, top row). Efforts to
produce antipeptide antisera to distinguish between the two isoforms proved
difficult. Therefore, to examine the targeting of these two OGT isoforms we
expressed myc-tagged versions of m OGT and ncOGT
(Fig. 2B). As we had previously
documented that expressing the full-length, catalytically active mOGT was
quite toxic (Lubas et al.,
1997
), we replaced the last 93 amino acids of the catalytic
domains of ncOGT and mOGT with myc tags. We have previously shown that this
deletion inactivates the catalytic activity of OGT
(Lubas and Hanover, 2000
).
Immunolocalization of myc-tagged ncOGT indicated a somewhat diffuse pattern
distributed throughout the nucleus and cytoplasm with no enrichment in
mitochondria (Fig. 2Ba). By
contrast, mOGT was associated with cytoplasmic structures highly suggestive of
mitochondria (Fig. 2Bb). This
localization was not eptitope-tag-specific; the same localization pattern was
observed when GFP was used instead of the myc epitope (data not shown). The
importance of the N-terminus of mOGT for this unique targeting was
demonstrated by deletional analysis. Removal of the first 15 amino acids of
mOGT (GFP-
mOGT) prevented concentration in mitochondria and resulted in
a primarily cytoplasmic localization, with a small amount found in the nucleus
(Fig. 2Bc).
|
The N-terminus of mOGT is important for mitochondrial targeting
To confirm that the mOGT isoform is uniquely targeted to mitochondria, we
colocalized myc-tagged mOGT with both endogenous OGT and with a
mitochondrion-selective probe (Fig.
2C). As shown in the top panels, the myc-tagged mOGT colocalizes
with the endogenous mitochondrial OGT, while showing little if any
colocalization with the endogenous, nuclear OGT
(Fig. 2Ca-c). The myc-tagged
mOGT also colocalized with MitoTracker® Red CM-H2XRos, directly
demonstrating mitochondrial targeting (Fig.
2Cd-f). Taken together, these data suggest that the unique
N-terminus of mOGT contains targeting information essential for mitochondrial
localization of mOGT.
O-GlcNAc-modified substrates do not accumulate in mitochondria
Although mOGT clearly localizes to mitochondria, biochemical and
morphological findings suggest that only low levels of O-GlcNAc-modified
substrates reside in mitochondria. As shown in
Fig. 3A, when endogenous
mitochondrial OGT (green) was colocalized with intracellular O-GlcNAc-modified
proteins (red), no colocalization (yellow) was observed in the mitochondria.
By contrast, nuclear OGT (Fig.
3A, green) and nuclear O-GlcNAc-modified proteins
(Fig. 3A, red) show many areas
of punctate localization. Another approach to visualizing the
O-GlcNAc-modified proteins was taken by probing HeLa subcellular fractions
with a monoclonal antibody (RL2) specific for O-GlcNAc-modified proteins
(Fig. 3B). Multiple proteins
ranging from 60 kDa to approximately 200 kDa were detected by RL2 and another
monoclonal antibody CTD 110.6 (data not shown). These proteins were
differentially enriched in the cytoplasm and nuclear fractions
(Fig. 3B, cyto, nuc). The
mitochondrial fraction, in comparison, was blank
(Fig. 3B, mito). Only extreme
overexposure of the western blot revealed very faint bands in the
mitochondrial fraction (data not shown). Previous reports have shown that
mitochondria contain the lowest amounts of cellular O-GlcNAc-modified proteins
(Hanover et al., 1987;
Holt and Hart, 1986
). Taken
together, these findings suggest that either mitochondrial OGT is
catalytically inactive in this organelle or the substrates for OGT are very
limited in mitochondria.
|
mOGT and catalytic activity
To demonstrate that the mOGT is capable of catalytic activity, we performed
two kinds of experiments. First, we showed that mOGT is catalytically active
when expressed in E. coli (Lubas
and Hanover, 2000). Second, we replaced the mitochondrial
targeting sequence of mOGT with GFP (GFP-
mOGT, see above) and expressed
the protein in HeLa cells (Fig.
3C). GFP-
mOGT is not targeted to mitochondria but resides
mainly in the cytoplasm, with trace amounts detected in the nucleus
(Fig. 3C, GFP-
mOGT).
Staining these transfected cells with RL2 showed enhanced glycosylation in the
cytoplasm of cells overexpressing GFP-
mOGT
(Fig. 3C, RL2). Taken together,
these studies argue that mitochondrial OGT, although potentially active, is
sequestered from the more abundant substrates found in the nucleus and
cytoplasm.
Association of mOGT with the mitochondrial membrane
To determine the mitochondrial localization of OGT at higher resolution,
purified HeLa cell mitochondria were examined by indirect immunogold electron
microscopy using affinity-purified anti-OGT
(Fig. 4A). We determined that
approximately 75% of the total gold particles were associated with the
mitochondrial membrane. Upon examining a large number of labeled mitochondria,
the majority of the gold label was associated with the mitochondrial inner
membrane (Fig. 4A). By
contrast, when a control incubation was performed
(Fig. 4A, negative control)
very little mitochondrial label was detected. Our morphometric analysis
suggested that, on average, 13.4 gold particles were associated with each
mitochondrion versus 0.64 gold particles per mitochondrion in the negative
control. These data suggest that OGT preferentially associates with the
mitochondrial inner membrane.
We used biochemical fractionation of isolated HeLa cell mitochondria and western blot analysis to corroborate the electron microscopy findings. HeLa mitochondria were fractionated into membrane-associated and soluble proteins by sonication and high-speed centrifugation (Fig. 4B). As we demonstrated in Fig. 1B, the 103 kDa mOGT species was enriched more than 10-fold in the mitochondria relative to the 116 kDa form (Fig. 4B, whole). Upon further fractionation of the mitochondria, mOGT was enriched in the salt-resistant membrane fraction (Fig. 4B, membrane). Little mOGT was present in the salt-extractable soluble fraction (Fig. 4B, soluble). The bottom row demonstrates that mitochondrial hsp70 (a matrix marker) is largely removed from the membrane fraction. To confirm the inner membrane localization, mitoplasts were made by hypotonic lysis of mitochondria. The localization of OGT was then compared in whole mitochondria and mitoplasts following a carbonate wash to remove proteins peripherally associated with the membrane (Fig. 4C). mOGT was found almost exclusively in the membrane fraction of both mitochondria and mitoplasts (Fig. 4C, arrows). Taken together, these data are consistent with the immunogold microscopic evidence, suggesting that mOGT is tightly associated with the mitochondrial inner membrane.
The sequence adjacent to the presumptive mitochondrial targeting of mOGT is
quite hydrophobic and is predicted to be a membrane-spanning region using a
number of algorithms (Fig. 2A,
underlined). The data presented here are consistent with a tight association
of the mOGT isoform to the mitochondrial inner membrane. Although the
algorithms used predict that mOGT is mainly oriented toward the intermembrane
space, further in vitro studies are required to confirm this localization.
These findings are intriguing in light of early reports from our laboratory
(Lubas et al., 1995;
Lubas et al., 1997
;
Starr and Hanover, 1990
) and
the Hart laboratory (Haltiwanger et al.,
1992
) suggesting that both soluble and membrane-bound OGT
activities were detected. This mitochondrially sequestered form of OGT (mOGT)
is likely to represent the membrane-bound form of OGT reported earlier.
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Discussion |
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The regulation of the differing OGT isoforms
Our recent analysis of the mouse, rat
(Hanover et al., 2002) and the
human (Nolte and Muller, 2002
)
ogt gene suggests that the isoforms we have designated mOGT and ncOGT
arise by alternative splicing from a single gene on the X-chromosome. The mOGT
form utilizes intron 4 of ncOGT as an exon to generate its unique 5'
N-terminus. Interestingly, this intronic region of the ogt gene may
also serve as an internal promoter driving the expression of only the mOGT
isoform. Thus, the current evidence suggests that the two isoforms of OGT may
be independently regulated. The mOGT and ncOGT isoforms differ in a number of
key respects. First, they differ in localization as we have demonstrated in
this study. Second, they differ in the number of TPR motifs present; mOGT has
nine repeats whereas the ncOGT has 12. Third, the unique N-termini of the two
proteins may contain additional regulatory information unrelated to targeting.
Given these differences, it is likely that the two OGT isoforms have very
different substrates and intracellular functions.
Functional significance of OGT isoforms
O-GlcNAc addition and removal is dynamic and exhibits a complex
interrelationship with protein phosphorylation. The O-GlcNAc modification has
been implicated in a large number of diverse intracellular processes ranging
from translational control, transcription, transcriptional repression, insulin
resistance and regulation of the cell cycle
(Hanover, 2001;
Wells et al., 2001
). These
functions may have as a common theme the requirement for nutrient sensing and
signaling through the hexosamine biosynthetic pathway (the hexosamine
signaling pathway) (Hanover,
2001
; Wells et al.,
2001
). Although the TPR domain of OGT is capable of interacting
with many proteins, it is difficult to understand how one enzyme derived from
a single gene could mediate such diverse intracellular functions. The model
shown in Fig. 5 summarizes how
the differentially targeted isoforms may contribute to hexosamine-dependent
intracellular signaling. Transcripts derived from a single mammalian OGT gene
on chromosome X are alternatively spliced to produce mOGT and ncOGT. The mOGT
and ncOGT isoforms are likely to perform unique functions in the mitochondrion
and nucleus in response to changing levels of a common precursor: UDP-GlcNAc.
The mitochondrial mOGT may participate in such functions as apoptosis and the
regulation of precursor transport and carbohydrate metabolism. The hexosamine
pathway has been previously implicated in the regulation of apoptosis
(Boehmelt et al., 2000
;
Hanover et al., 1999
;
Liu et al., 2000
). In
addition, we have shown that when overexpressed, the mOGT is highly toxic to
cells (Lubas et al., 1997
). By
contrast, overexpression of ncOGT is not overtly cytotoxic
(Kreppel et al., 1997
)
(J.A.H., unpublished). A clear relationship also exists between mitochondrial
superoxide overproduction and O-GlcNAc metabolism
(Du et al., 2000
;
Du et al., 2001
;
Nishikawa et al., 2000
). The
mOGT isoform is, therefore, likely to participate in the hexosamine-dependent
changes in mitochondrial function in response to lipid and carbohydrate
metabolism or proapoptotic signals.
|
The unique N-terminus, additional TPRs and nucleocytoplasmic localization
of ncOGT suggests that this isoform participates in the cytoplasmic and
nuclear events attributed to O-GlcNAc such as translation, nuclear transport,
transcriptional repression and chromatin remodeling
(Datta et al., 1989;
Hanover, 2001
;
Kelly and Hart, 1989
;
Wells et al., 2001
). Most
recently, ncOGT was shown to interact with the histone deacetylation complex
by binding to the corepressor mSin3a (Yang
et al., 2002
). By appropriate targeting of ncOGT by mSin3a,
transcription can be negatively regulated through glycosylation and
deacetylation. Therefore, the identification and characterization of the
differentially targeted isoforms of OGT presented here should facilitate
further dissection of the regulation of their unique intracellular
functions.
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Acknowledgments |
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References |
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