Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

In an initial construct, the Tac lumenal domain was fused to the transmembrane and cytoplasmic domains of TGN38 to create the chimeric protein, T-G-G (.T_ac lumenal-T__GN38 transmembrane-TG_N38 cytoplasmic) (Fig. 1, top) . Plasmids encoding Tac and T-G-G were transfected separately into the rat fibroblast line, NRK, and the subeellular distribution of both proteins was examined by immunofluorescence microscopy of fixed, permeabilized cells using an antibody to a Tac lumenal epitope (7G7) (Rubin et al., 1985a) . We observed that whereas normal Tac was expressed on the plasma membrane ( Fig. 1 a) , the T-G-G chimera was localized to a juxtanuclear, tubulo-vesicular structure in the majority of positively stained cells (Fig. 1 c) . This distribution was consistent with localization to the TGN, as demonstrated by the similar pattern of immunofluorescent staining observed for endogenous TGN38 in the same cells ( Fig. 1 d) . Approximately 90% of the positively stained NRK cells showed localization of the chimeric protein to the TGN, whereas the remaining 10% also showed localization to the plasma membrane. In addition, ,,o15 % of the cells displayed staining of cytoplasmic vesicles. No changes in the localization of the T-G-G chimera were noticed upon a 3-h incubation of the cells in 10/~g/ml cycloheximide, suggesting that the localization of the protein to the TGN was not a transient phenomenon. In addition to NRK cells, the localization of T-G-G was examined after transient expression in various other cell lines, including cells of monkey (CV-1, COS-l), bovine (MBDK), and human origins (RD4 and HeLa). In all cases, we observed prominent staining of a tubulo-vesicular, and sometimes distinctly reticular, structure characteristic of the TGN (data not shown). These observations suggested that the TGN localization of proteins containing sequences from TGN38 was conserved among several mammalian species. A variable proportion of the cells also showed staining of cytoplasmic vesicles and the plasma membrane, the appearance of which seemed to correlate with higher levels of expression in those cells.