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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