NBD‐Labeled Phosphatidylcholine and Phosphatidylethanolamine are Internalized by Transbilayer Transport across the Yeast Plasma Membrane

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Abstract
The internalization and distribution of fluorescent analogs of phosphatidylcholine (M‐C6‐NBD‐PC) and phosphatidylethanolamine (M‐C6‐NBD‐PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M‐C6‐NBD‐PC was internalized predominantly to the vacuole and degraded. M‐C6‐NBD‐PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2°C, both were internalized to the nuclear envelope/ER and mitochondria by an energy‐dependent, N‐ethylmaleimide‐sensitive process, and transport of M‐C6‐NBD‐PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis‐defective mutant, end4‐1. However, following pre‐incubation at 37°C, internalization of both phospholipids was inhibited at 2°C and 37°C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M‐C6‐NBD‐PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M‐C6‐NBD‐PC and M‐C6‐NBD‐PE are transported across the plasma membrane to the cytosolic leaflet by a protein‐mediated, energy‐dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M‐C6‐NBD‐PC, but not M‐C6‐NBD‐PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

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