Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase.
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.
Love
Dona C
DC
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
Kochan
Jarema
J
Cathey
R Lamont
RL
Shin
Sang-Hoon
SH
Hanover
John A
JA
Kochran
Jarema
J
D017398:Alternative Splicing / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology*; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D003593:Cytoplasm / Q000201:enzymology* / Q000648:ultrastructure; D005057:Eukaryotic Cells / Q000201:enzymology* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D011134:Polysaccharides; D020033:Protein Isoforms; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase