Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells.
In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.
Baumann
N A
NA
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Vidugiriene
J
J
Machamer
C E
CE
Menon
A K
AK
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000737:chemistry; D013696:Temperature; D013945:Thymoma / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014780:Viruses / Q000737:chemistry
D017261:Glycosylphosphatidylinositols; D020126:Brefeldin A; D008358:Mannose