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First published online 18 December 2002
doi: 10.1242/jcs.00246

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Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Dona C. Love¹, Jarema Kochran², R. Lamont Cathey^1,*, Sang-Hoon Shin¹ and John A. Hanover^1,

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

View larger version (88K): [in a new window]   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.

View larger version (65K): [in a new window]   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.

View larger version (87K): [in a new window]   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.

View larger version (73K): [in a new window]   Fig. 3. O-GlcNAc-modified glycoproteins do not accumulate in mitochondria although mOGT is catalytically active. (A) Untransfected HeLa cells (upper row) were grown on glass coverslips, fixed and processed for indirect immunofluorescence to reveal endogenous OGT (OGT) and O-GlcNAc (RL2) localization. The two images were overlaid to determine colocalization (Merge). (B) HeLa subcellular fractions were probed for the O-GlcNAc-modified proteins. Equal amounts of total HeLa lysate (total), cytosol (cyto), nuclear (nuc) and mitochondrial proteins were separated using a 10% Bis-Tris SDS gel, transferred to nitrocellulose and probed with the RL2 monoclonal antibody. The migration of molecular weight markers (kDa) is shown on the left. (C) To demonstrate that mOGT is catalytically active when mislocalized from mitochondria to the cytoplasm, HeLa cells were transfected with GFP-mOGT for 24 hours, fixed and processed to visualize GFP-mOGT. Indirect immunofluorescence was used to localize O-GlcNAc (RL2). Bars, 10 µM.

View larger version (27K): [in a new window]   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.