Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking

The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but it remains unclear how the cell discerns among the different ceramides to affect such sorting. To address the mechanism, we synthesize a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assess their sorting among different endocytic pathways. We find that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictate lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14* motif is cholesterol dependent. Perturbations of the C14* motif by unsaturation enable GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independent of cholesterol. Unsaturation occurring beyond the C14* motif in very long acyl chains rescues lysosomal sorting. These results define a structural motif underlying the membrane organization of sphingolipids and implicate cholesterol-sphingolipid nano-domain formation in sorting mechanisms.