Here we report the existence of two OGT isoforms differing in their subcellular localization. The shorter form associates with mitochondria in both primary and immortilized cells, whereas the longer form is nucleocytoplasmic. The N-terminus of the short form contains mitochondrial targeting information and is essential for mitochondrial localization. These data are discussed in terms of the role of differential targeting of OGT to the mitochondrion and nucleus in the regulation of a hexosamine-dependent signaling pathway.
Materials and Methods
Plasmids pmOGT-myc was created by inserting a PCR product containing a BamHI site, Kozac consensus sequence and nucleotides spanning the atg start site to the XhoI site of mogt into pcDNA4/myc-His C (Invitrogen, Carlsbad, CA). pcnOGT-myc was created by digestion of pLKG1 (Kreppel et al., 1997), containing full-length rat cnOGT, with HindIII and XhoI. This purified fragment was inserted into pCDNA4myc His C (Invitrogen) using the same sites. Finally, pGFP-mOGT was created by digesting pLV4f (Lubas et al., 1997), encoding full-length mOGT, with EcoRV and BamHI and inserting the purified fragment into the EcoICR1 and BamHI sites of pEGFP-C2 (Invitrogen).
Transfections
HeLa cells were seeded on glass coverslips in six-well plates (Corning Incorporated, Corning, NY) at 1×10 5 cells per well. Once cells were attached to the coverslips, they were transfected with indicated plasmids, using Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instructions.

### Language ###
English

### Assessment ###
Could ask about the specific tool used for cell transfection in this protocol

### Query ###
For cell transfection experiments, tell me what reagent Fugene 6 is manufactured by.
For cell transfection experiments, tell me what reagent Fugene 6 is manufactured by.