NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane.
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 degrees 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 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees 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.
Grant
A M
AM
Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Hanson
P K
PK
Malone
L
L
Nichols
J W
JW
D000251:Adenosine Triphosphatases; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008051:Lipid Bilayers; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D019810:Sodium Azide / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013696:Temperature; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D004791:Enzyme Inhibitors; C092350:FM 4-64; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D012969:Sodium Fluoride; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D005033:Ethylmaleimide