Transport of a Fluorescent Phosphatidylcholine Analog from the Plasma Membrane to the Golgi Apparatus

• Published in: The Journal of cell biology 1984-08, Vol.99 (2), p.742-751
• Main Authors: Sleight, Richard G., Pagano, Richard E.
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - metabolism; Animals; B lymphocytes; Biological and medical sciences; Biological Transport; Cell Line; Cell Membrane - metabolism; Cell membranes; Cell physiology; Cells; Cricetinae; Cricetulus; Cultured cells; Endocytosis; Fluorescence; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; Golgi apparatus; Golgi Apparatus - metabolism; Internalization; Kinetics; Lipid Bilayers; Lipids; Lung; Membrane and intracellular transports; Microscopy, Fluorescence; Molecular and cellular biology; P branes; Phosphatidylcholines - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.99.2.742

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2°C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2°C; however, when the cells were warmed to 37°C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37°C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37°C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2°C and neither were internalized at 37°C in the presence of inhibitors of endocytosis. However, when incubated at 37°C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.