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J. Biol. Chem., Vol. 277, Issue 36, 33325-33333, September 6, 2002
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From the Departments of The major pathway for cellular uptake of
the water-soluble vitamin folic acid in mammalian cells is via a plasma
membrane protein known as the reduced folate carrier (RFC). The
molecular determinants that dictate plasma membrane expression of RFC
as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to
image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the
polypeptide and not within the cytoplasmic NH2- or
COOH-terminal domains of the protein. Further, the integrity of the
hRFC backbone is critical for export of the polypeptide from the
endoplasmic reticulum to the cell surface. This trafficking is
critically dependent on intact microtubules because microtubule
disruption inhibits motility of hRFC-containing vesicles as well as
final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.
Intracellular Trafficking and Membrane Targeting
Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial
Cells*,
§¶,
**,, and**¶¶
Neurobiology and Behavior,
Medicine, and ** Physiology and Biophysics, University
of California, Irvine, California 92697, the § Department of
Pharmacology, University of Minnesota, 55455, and the
Department of Veterans Affairs Medical
Center, Long Beach, California 90822
*
This work was supported by the Department of Veterans
Affairs, the University of Minnesota Medical School, and National
Institutes of Health Grants DK-56061 and DK-58057 (to H. M. S.) and
GM-48071 (to I. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at www.jbc.org)
contains Videos 1-4.
¶
Both authors contributed equally to this work.
¶¶
To whom correspondence should be addressed.
Tel.: 562-826-5811; Fax: 562-826-5731; E-mail: hmsaid@uci.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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