Abstract
Photoactivatable derivatives of ceramide, glucosylceramide (GlcCer) and sphingomyelin {3-(p-azido-m-[125I]iodophenyl)propionylceramide, 3-(p-azido-m-[125I]iodophenyl)propionyl-GlcCer and 3-(p-azido-m-[125I]iodophenyl)propionylsphingomyelin} were synthesized in an attempt to identify compartment-specific proteins involved in sphingolipid sorting or metabolism. In HT29 and BHK cells the ceramide analogue entered the cell by monomeric diffusion, as evidenced by the probe's efficient internalization at low temperature (4 degrees C). In contrast, the photoactivatable GlcCer was internalized only at elevated temperatures (37 degrees C), presumably reflecting an endocytic mechanism of uptake. The photoactivatable ceramide was mainly metabolized to the corresponding sphingomyelin analogue, but small amounts of GlcCer and galactosylceramide were also synthesized. The newly synthesized photoreactive sphingomyelin was subsequently transported to the cell surface, a process that was effectively inhibited by the presence of brefeldin A. The incubation of cells with photoactivatable analogues at 4 degrees C, followed by illumination, led to the association of sphingolipid with a specific subset of proteins. The protein labelling pattern of ceramide differed from that of glucosylceramide. A further shift in labelling pattern was apparent when the cells were incubated with the lipid analogues at 37 degrees C. Moreover, most of the proteins labelled by photoreactive sphingomyelin seemed to be detergent-insoluble, which is indicative of a location in sphingolipid-rich microdomains at the plasma membrane. The potential of applying photoactivatable sphingolipids to further define and identify the role of distinct proteins in sphingolipid biosynthesis, transport and sorting, is discussed.