An asymmetric distribution of phospholipids between the leaflets
of the plasma membrane has been observed to be an ubiquitous property of
eukaryotes. The aminophospholipids, phosphatidylethanolamine and
phosphatidylserine are sequestered on the inner leaflet of the plasma
membrane and the majority of the phosphatidylcholine is located on the
external leaflet. Work from our lab has demonstrated the efficacy of
using the yeast, S. cerevisiae , to study retrograde transport of
phospholipids using the fluorescent phosphatidylethanolamine and
phosphatidylcholine analogs, NBD-PE and NBD-PC. Using a combination of
fluorescence microscopy and flow cytometry, we have observed that
sec mutants defective in the vesicular trafficking steps of
secretion were defective in the accumulation of NBD-PE and NBD-PC, but
not the endocytic dye, FM 4-64. In addition, act1-1 and end4-1 mutants, which are defective in alpha factor internalization, were
defective in FM 4-64 accumulation but were not defective in the
accumulation of NBD-PE and NBD-PC. Once in the cell, NBD-PE localized to
the mitochondria, nuclear envelope and ER. NBD-PC localized to these
organelles as well but was also sorted to the vacuole and sorting to the
vacuole was inhibited in vps4 and vps28 null strains.
These data suggest that both NBD-PE and NBD-PC are internalized by
transmembrane translocation across the plasma membrane and distributed
to the mitochondria nuclear envelope and ER and that NBD-PC, but not
NBD-PE, is transported to the vacuole via the prevacuolar intermediate
compartment. Supported by NIH grant GM52410 and NIH minority fellowship
GM18012-03.
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