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A more recent version of this article appeared on February 1, 2004
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Submitted on July 16, 2003
Revised on September 26, 2003
Accepted on September 27, 2003
1 Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., Rm. E-215, New York, NY 10021, These authors contribute equally to this work
2 Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., Rm. E-215, New York, NY 10021, Department of Molecular Oncology, Genentech, Inc, 1 DNA Way, MS 42, South San Francisco, CA 94080, These authors contribute equally to this work
3 Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., Rm. E-215, New York, NY 10021
* Corresponding author. E-mail address: frmaxfie{at}med.cornell.edu.
Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. Following endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment (ERC). A large fraction of the receptors return to the plasma membrane, but some are delivered to the TGN and/or late endosomes. Over the course of an hour the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the ERC. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.
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