USE OF PHOTOACTIVATABLE SPHINGOLIPID ANALOGS TO MONITOR LIPID TRANSPORT IN MAMMALIAN-CELLS

Photoactivatable derivatives of ceramide, glucosylceramide (GlcCer) an d sphingomyelin {3-(p-azido-m-[I-125]iodophenyl)propionylceramide, 3-( p-azido-m-[I-125]iodophenyl)propionyl-GlcCer and -azido-m-[I-125]iodop henyl)propionylsphingomyelin} were synthesized in an attempt to identi fy compartment-specific proteins involved in sphingolipid sorting or m etabolism. In HT29 and BHK cells the ceramide analogue entered the cel l by monomeric diffusion, as evidenced by the prober's efficient inter nalization at low temperature (4 degrees C). In contrast, the photoact ivatable GlcCer was internalized only at elevated temperatures (37 deg rees C), presumably reflecting an endocytic mechanism of uptake. The p hotoactivatable ceramide was mainly metabolized to the corresponding s phingomyelin analogue, but small amounts of GlcCer and galactosylceram ide were also synthesized. The newly synthesized photoreactive sphingo myelin was subsequently transported to the cell surface, a process tha t was effectively inhibited by the presence of brefeldin A. The incuba tion of cells with photoactivatable analogues at 4 degrees C, followed by illumination, led to the association of sphingolipid with a specif ic subset of proteins. The protein labelling pattern of ceramide diffe red from that of glucosylceramide. A further shift in labelling patter n was apparent when the cells were incubated with the lipid analogues at 37 degrees C. Moreover, most of the proteins labelled by photoreact ive sphingomyelin seemed to be detergent-insoluble, which is indicativ e of a location in sphingolipid-rich micro-domains the plasma membrane . The potential of applying photoactivatable sphingolipids to further define and identify the role of distinct proteins in sphingolipid bios ynthesis, transport and sorting, is discussed.