Transport of sphingolipids by yeast Npc2 supports phase separation of the vacuole membrane.

The yeast vacuole membrane forms ordered microdomains that facilitate microlipophagy under nutrient limitation. We previously found that this process involves the intracellular sorting of sphingolipids to the vacuole. While multiple vacuole protein pathways have been identified, corresponding mechanisms for lipid sorting remain undefined. Here, we use a range of approaches to identify how endocytic sorting and intraluminal transport of sphingolipids contribute to the formation of vacuole domains. To visualize sphingolipid trafficking, we employed the ceramide (Cer) analog BODIPY C12-ceramide (BODIPY-Cer), which is internalized by cells and stains the vacuole. We observed that cells lacking Vps29 and Vps30, proteins involved in endosomal sorting, show altered vacuole domains and accumulate BODIPY-Cer at sites proximal to the plasma membrane. Subsequent incorporation of endocytic-derived Cer into the vacuole is dependent on the Niemann-Pick type C2 protein (Npc2). Loss of Npc2 reduces domain formation and causes BODIPY-Cer to accumulate within the vacuole lumen. Both intravacuole trafficking of BODIPY-Cer and membrane phase separation were not dependent on Npc2's canonical receptor, Ncr1. Lipidomics of isolated vacuoles confirmed that Npc2 independently mediates sphingolipid sorting under microlipophagy conditions. In liposome assays, yeast Npc2-but not its human homolog-robustly transports an analog of inositol phosphorylceramide, a complex sphingolipid that is enriched in phase-separated vacuoles. We propose that the enlarged binding cavity of yeast Npc2 is specialized for the incorporation of sphingolipids into the vacuole membrane to support its phase separation.