Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by...

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.