Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Abstract

O-linked GlcNAc transferase (OGT) mediates a novel glycan-dependent signaling pathway, but the intracellular targeting of OGT is poorly understood. We examined the localization of OGT by immunofluorescence microscopy, subcellular fractionation and immunoblotting using highly specific affinity-purified antisera. In addition to the expected nuclear localization,we found that OGT was highly concentrated in mitochondria. Since the mitochondrial OGT (103 kDa) was smaller than OGT found in other compartments(116 kDa) we reasoned that it was one of two predicted splice variants of OGT. The N-termini of these isoforms are unique; the shorter form contains a potential mitochondrial targeting sequence. We found that when epitope-tagged,the shorter form (mOGT; 103 kDa) concentrated in HeLa cell mitochondria,whereas the longer form (ncOGT; 116 kDa) localized to the nucleus and cytoplasm. The N-terminus of mOGT was essential for proper targeting. Although mOGT appears to be an active transferase, O-linked GlcNAc-modified substrates do not accumulate in mitochondria. Using immunoelectron microscopy and mitochondrial fractionation, we found that mOGT was tightly associated with the mitochondrial inner membrane. The differential localization of mitochondrial and nucleocytoplasmic isoforms of OGT suggests that they perform unique intracellular functions.

O-GlcNAc and the Epigenetic Regulation of Gene Expression

“…O-GlcNAc is added by the enzyme OGT to serine and threonine of target proteins in the cytoplasm, nucleus, and mitochondrion (27,28). The single mammalian OGT gene is located on the human X chromosome near the Xist locus and encodes multiple alternatively spliced isoforms of OGT targeted to these different intracellular locations (27,28 isoforms are termed mOGT (mitochondrial OGT), ncOGT (nucleocytoplasmic OGT), and sOGT (short OGT) (Fig.…”

“…1B). The mOGT isoform is targeted to the mitochondrial matrix and is proapoptotic (28,29). The sOGT isoform, present in the nucleus and cytoplasm, is antiapoptotic (28,30) and may inhibit the action of the other isoforms (29,31).…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…Unlike many other glycosyltransferases, OGT is a soluble protein that is predominantly localized to the nucleus, mitochondria, and cytoplasm of all tissues studied thus far (Kreppel et al 1997;Haltiwanger et al 1992;Lubas et al 1997;Love et al 2003;Hanover et al 2003). Two functional domains characterize OGT: an N-terminal tetratricopeptide repeat (TPR domain) and a C-terminal catalytic domain belonging to the glycogen phosphorylase superfamily (Kreppel et al 1997;Lubas et al 1997;Wrabl and Grishin 2001).…”

“…The gene for OGT maps to Xq13 on the mammalian X chromosome and encodes three well-characterized variants: short OGT, mitochondrial OGT (mOGT), and nuclear/cytoplasmic OGT (Kreppel et al 1997;Lubas et al 1997;Love et al 2003;Hanover et al 2003). mOGT has an alternative mitochondrial targeting sequence on its N-terminus.…”

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis

“…Impaired content or activity of enzymes related to mitochondrial function has been reported in STZ-or HFD-induced diabetic animal models [20][21][22][23]. Following the observation of OGT localisation to the mitochondria by Love et al [24], the presence of OGA was recently reported in cardiac mitochondria in rats [25]. Despite a recently identified association between OGA-mediated O-GlcNAcylation and diabetes progression [25,26], there have been no reports of the effects of OGA activity on mitochondrial regulation.…”