Brefeldin A does not inhibit the movement of phosphatidylethanolamine from its sites for synthesis to the cell surface.
Brefeldin A, a fungal metabolite which interrupts trafficking of proteins via the Golgi by causing disassembly of the Golgi stacks, has been used to investigate the mechanism of movement of phosphatidylethanolamine (PtdEtn) from its sites of synthesis to the cell surface. PtdEtn is made in hepatocytes by two major pathways, (a) from CDP-ethanolamine on the endoplasmic reticulum and (b) by decarboxylation of phosphatidylserine in mitochondria. Monolayer cultures of rat hepatocytes were incubated with radiolabeled precursors of PtdEtn ([3H]ethanolamine or [3H]serine) in the presence or absence of brefeldin A. The movement of newly made PtdEtn to the plasma membrane was studied by treatment of intact cells with trinitrobenzene sulfonate which reacted only with PtdEtn on the outside surface of the cells to produce N-trinitrophenyl-PtdEtn; PtdEtn in intracellular membranes remained underivatized by this reagent. Using this method, the incorporation of radioactivity into cell surface and intracellular PtdEtn could be differentiated. The studies showed that PtdEtn made by the two different biosynthetic routes was rapidly transported to the outside leaflet of the plasma membrane. However, the kinetics and the extent of labeling of the cell surface PtdEtn, relative to that of the intracellular PtdEtn pool, were different from each labeled precursor. The incorporation of [3H]ethanolamine into PtdEtn on the cell surface gradually increased to a constant level of 1.8% of the label of intracellular PtdEtn after 3 h. In contrast, after 0.5-1 h, cell surface PtdEtn labeled from [3H]serine comprised 4% of the intracellular PtdEtn pool; the extent of labeling gradually declined to a constant level of approximately 2.4% by 3 h. Brefeldin A did not interrupt the movement of PtdEtn, derived from either biosynthetic origin, to the cell surface even though protein secretion was greatly reduced. Thus, apparently PtdEtn and proteins are independently transported to the cell surface of hepatocytes.
Vance
J E
JE
Lipid and Lipoprotein Group, University of Alberta, Edmonton, Canada.
Aasman
E J
EJ
Szarka
R
R
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology*; D005260:Female; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D003517:Cyclopentanes; D010714:Phosphatidylethanolamines; D020126:Brefeldin A