Sorting and Intracellular Trafficking of a Glycosylphosphatidylinositol-anchored Protein and Two Hybrid Transmembrane Proteins with the Same Ectodomain in Madin-Darby Canine Kidney Epithelial Cells (*)
- From theDepartment of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215
- § To whom correspondence should be addressed. Tel.: 516-632-8563; Fax: 516-632-8575.
Abstract
We compared the trafficking of the glycosylphosphatidylinositol (GPI)-anchored placental alkaline phosphatase (PLAP) and two chimeric transmembrane proteins containing the PLAP ectodomain in stably transfected Madin-Darby canine kidney epithelial cells to determine whether different mechanisms might be used in apical sorting of GPI-anchored and transmembrane proteins. PLAP-G, which contained the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein, was delivered directly to the basolateral surface. PLAP-HA contained the transmembrane and cytoplasmic domains of influenza hemagglutinin. Both PLAP and PLAP-HA were delivered directly to the apical membrane. PLAP becomes insoluble in Triton X-100 during biosynthetic transport, as it associates with detergent-resistant membranes. Neither hybrid protein was detergent insoluble, though the small amount of PLAP that was missorted to the basolateral surface was insoluble. We examined the effects of three drugs known to interfere with membrane trafficking on sorting and delivery of PLAP and the hybrid proteins. Monensin had no effect on sorting or surface expression of any of the proteins. Nocodazole affected the sorting of both PLAP and PLAP-HA but not of PLAP-G. Brefeldin A appeared to disrupt the sorting of PLAP and PLAP-HA but not of PLAP-G. This conclusion was tempered by the observation that this drug affected the distribution of proteins at the cell surface. Thus, sorting and transport of GPI-anchored and apical transmembrane proteins are similar in a number of respects.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM47897 (to D. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵(1) The abbreviations used are:
- MDCK
-
Madin-Darby canine kidney
- HA
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hemagglutinin
- PLAP
-
placental alkaline phosphatase
- GPI
-
glycosylphosphatidylinositol
- VSV G
-
vesicular stomatitis virus glycoprotein
- PAGE
-
polyacrylamide gel electrophoresis
- PBS
-
phosphate-buffered saline
- BFA
-
brefeldin A
- PI-PLC
-
phosphatidylinositol-specific phospholipase C
- TGN
-
trans-Golgi network
- FRT
-
Fischer rat thyroid.
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↵(2)K. Melkonian and D. Brown, unpublished data.
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- Received March 22, 1995.
- Revision received July 21, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
