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J. Cell Biol.,
Volume 142, Number 4, August 24, 1998 923-936
Department of Biochemistry, Cornell University Medical College, New York, New York 10021
To examine TGN38 trafficking from the cell
surface to the TGN, CHO cells were stably transfected
with a chimeric transmembrane protein, TacTGN38.
We used fluorescent and 125I-labeled anti-Tac IgG and
Fab fragments to follow TacTGN38's postendocytic
trafficking. At steady-state, anti-Tac was mainly in the
TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38
is on the plasma 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 (t1/2 = 9 min), and the remainder 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 with a t1/2 of 46 min.
Quantitative confocal microscopy analysis also showed
that fluorescent anti-Tac fills the TGN with a 46-min
t1/2. Using 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 distribution despite the inefficient targeting from the cell
surface to the TGN.
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