NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment.
At low temperature, the short-chain fluorescent-labeled phospholipids, 1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37 degrees C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole. Sorting of M-C6-NBD-PC to the vacuole is blocked by energy-depletion and by null mutations in the VPS4 and VPS28 genes required for vesicular traffic from the pre-vacuolar compartment (PVC) to the vacuole. This sorting is not blocked by a temperature-sensitive mutation in SEC12, which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane. Monomethylation or dimethylation of the primary amine head-group of M-C6-NBD-PE is sufficient for sorting to the yeast vacuole in both wild-type yeast and in strains defective in the phosphatidylethanolamine methylation pathway. These data indicate that methylation of M-C6-NBD-PE produces the crucial structural component required to sort these phospholipid analogues to the vacuole via the PVC.
Hanson
Pamela K
PK
Department of Physiology, 615 Michael St, 605G Whitehead Building, Emory University School of Medicine, Atlanta, GA 30322, USA.
Grant
Althea M
AM
Nichols
J Wylie
JW
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008745:Methylation; D009154:Mutation / Q000502:physiology; D010713:Phosphatidylcholines / Q000378:metabolism* / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D009327:4-Chloro-7-nitrobenzofurazan