Rn. Ghosh et al., AN ENDOCYTOSED TGN38 CHIMERIC PROTEIN IS DELIVERED TO THE TGN AFTER TRAFFICKING THROUGH THE ENDOCYTIC RECYCLING COMPARTMENT IN CHO CELLS, The Journal of cell biology, 142(4), 1998, pp. 923-936
To examine TGN38 trafficking from the cell surface to the TGN, CHO cel
ls were stably transfected with a chimeric transmembrane protein, TacT
GN38. We used fluorescent and I-125-labeled anti-Tac IgG and Fab fragm
ents to follow TacTGN38's postendocytic trafficking. At steady-state,
anti-Tac was mainly in the TGN, but shortly after endocytosis it was p
redominantly in early endosomes, 11% of cellular TacTGN38 is on the pl
asma membrane. Kinetic analysis of trafficking of antibodies bound to
TacTGN38 showed that after short endocytic pulses, 80% of internalized
anti-Tac returned to the cell surface (t(1/2) = 9 min), and the remai
nder trafficked to the TGN. When longer filling pulses and chases were
used to load anti-Tac into the TGN, it returned to the cell surface w
ith a t(1/2) Of 46 min. Quantitative confocal microscopy analysis also
showed that fluorescent anti-Tac fills the TGN with a 46-min t(1/2) U
sing the measured rate constants in a simple kinetic model, we predict
that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38
leaves the TGN slowly, which accounts for its steady-state distributio
n despite the inefficient targeting from the cell surface to the TGN.