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© The Rockefeller University Press, 0021-9525/1998//923 $5.00
The Journal of Cell Biology, Volume 142, Number 4, , 1998 923-936

Articles

An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells



Richik N. Ghosh, William G. Mallet, Thwe T. Soe, Timothy E. McGraw, and Frederick R. Maxfield

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.

Key Words: trans-Golgi network • endocytosis • endosome • protein sorting

Abbreviations used in this paper: ERC, endocytic recycling compartment; F-anti-Tac, fluorescein-conjugated anti-Tac antibody; F-IgG, fluorescein-conjugated IgG; F-Tf, fluorescein-conjugated transferrin; F-dex, fluorescein-conjugated dextran; M6PR, mannose-6-phosphate receptor; C6-NBD-ceramide, N-({varepsilon}-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)- D-erythro-sphingosine; R-dex, rhodamine-conjugated dextran; Tf, human transferrin; TR, transferrin receptor.

The first two authors contributed equally to this work.

Address all correspondence to Dr. F.R. Maxfield, Department of Biochemistry, Cornell University Medical College, New York, NY 10021. Tel.: 212-746-6405. Fax: 212-746-8875. E-mail: frmaxfie{at}mail.med.cornell.edu


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