First published online 18 December 2002
doi: 10.1242/jcs.00246
Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase
Dona C. Love1,
Jarema Kochran2,
R. Lamont Cathey1,*,
Sang-Hoon Shin1 and
John A. Hanover1,
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
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Fig. 1. Nucleocytoplasmic and mitochondrial localization of OGT. (A) HeLa cells
(top row) and HAEC (bottom row) were grown on glass coverslips, fixed and
processed for indirect immunofluorescence using affinity-purified anti-OGT
antibodies and a mouse monoclonal antibody raised against cytochrome C. The
confocal images representing OGT (green) and cytochrome C (red) were overlayed
(merge) to reveal colocalization. Bar, 10 µM. (B) Total cellular lysate
(total), nuclear and mitochondrial (mito) fractions from HeLa cells were
separated using a 10% PAGE-gel, transferred to nitrocellulose and probed with
OGT-specific, affinity-purified antibodies as described in Materials and
Methods. Equal amounts of protein were loaded in each lane. Note the
enrichment ( 10-fold) of the 103 kDa species in mitochondria.
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Fig. 4. OGT is associated with the mitochondrial inner membrane. (A) Immunogold
staining of purified mitochondria demonstrates an inner membrane localization
for OGT; no specific label was detected when the gold-labeled secondary
antibody was used alone (negative control). Bars, 0.1 µM. (B) Purified HeLa
mitochondria (whole) were fractionated by sonication and high-speed
centrifugation into membrane and soluble protein fractions, separated on a
4-20% PAGE-gel, transferred to nitrocellulose and probed with OGT-specific,
affinity-purified antibodies. Equal amounts of protein were loaded in each
lane. The majority of mOGT was found associated with the membrane fraction
after salt washing (see Materials and Methods). Fractionation of mitochondria
was monitored by mtHSP 70 content. The bulk of this matrix marker appeared in
the soluble fraction under conditions where mOGT remained membrane associated.
(C) Localization of mOGT was compared in mitochondria and mitoplasts subjected
to alkaline extraction with carbonate buffer. The outer membrane of
mitochondria was removed by hypotonic lysis to produce the mitoplasts. Both
mitochondria and the mitoplasts were washed with 100 mM sodium carbonate pH 11
to remove peripherally associated proteins. The soluble and membrane fractions
were separated and probed for OGT. The arrows indicate that the mOGT is
greatly enriched in the membrane fractions of both mitochondria and
mitoplasts.
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Fig. 2. Identification of nucleocytoplasmic and mitochondrial isoforms of OGT. (A)
A schematic of known OGT isoforms is shown to illustrate the unique N-termini.
ncOGT (top row) contains 89 amino acids and an additional three TPRs, compared
to mOGT (bottom row). mOGT contains a putative mitochondrial targeting
sequence at its N-terminus (shown in red) and a predicted membrane-spanning
helix (underlined). Both OGT isoforms are identical from the nine TPR region
to their C-termini. (B) The 116 kDa (ncOGT) and 103 kDa (mOGT) isoforms of OGT
were expressed in HeLa cells as a C-terminus myc-fusion. 24 hours following
transfection cells were fixed and processed for indirect immunofluorescence
using an anti-myc monoclonal antibody (a and b). ncOGT was distributed between
the nucleus and cytoplasm and did not accumulate in mitochondria (Ba). By
contrast, mOGT concentrated in cytoplasmic structures highly reminiscent of
mitochondria (Bb; see below). The third panel (Bc) shows the cytoplasmic
localization of GFP- mOGT, which lacks the mitochondrial targeting
region of mOGT. (C) The myc-tagged mOGT (Ca) colocalized with anti-OGT
antibodies (Cb), which recognize both OGT isoforms. Colocalization was
apparent only in the mitochondria; the tagged mOGT did not significantly
colocalize with the endogenous nuclear OGT (Cc). The lower panels (Cd-f) show
colocalization of mOGT (Cd) with MitoTracker® Red CM-H2XRos
(Ce), a mitochondrial marker. The merged image shows colocalization in
mitochondria (Cf). Bars, 10 µM.
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Fig. 5. A model depicting the role of differentially targeted OGT isoforms in
glycan-dependent signaling. The studies described here suggest that
transcription from the OGT gene and subsequent alternative splicing produces a
number of isoforms including mOGT and ncOGT. The unique N-termini of these
isoforms lead to segregation of mOGT into the mitochondrial membrane and
targeting of ncOGT to the nucleus and cytoplasm. The hexosamine-signaling
pathway is known to modulate the levels of UDP-GlcNAc in response to nutrients
(glucose, glutamine, free fatty acids). The UDP-GlcNAc would then be used by
each of the uniquely targeted OGT isoforms to modify different intracellular
substrates. In the mitochondrion, mOGT may play a role in apoptosis and lipid
and carbohydrate metabolism (see text). In the nucleus and cytosol, ncOGT is
envisioned to mediate effects on translation, nuclear transport and
transcriptional repression.
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© The Company of Biologists Ltd 2003
