Transbilayer Phosphatidylethanolamine Movements in the Yeast Plasma Membrane

Abstract

Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP-dependent, protein-mediated process. To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharornyces cerevisiae (A1 84D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes. The levels of external PtdEtn in the intact cells were reduced to about 50 % by pretreatment of the cells with inhibitors of mitochondria1 ATP synthesis, ATPase inhibitors or protein-sulfhydryl-group-modifying reagents, or by depletion of the cells of ATP by metabolic starvation. The levels of external PtdEtn could be restored to normal by repletion of the energy-depleted cells with ATP. Furthermore, treatment of the energy-depleted cells with sulfhydryl-modyfying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells. These results demonstrate an involvement of an ATP-dependent, protein-mediated process(es) in the regulation of the PtdEtn distribution across the plasma-membrane bilayer of yeast cells. The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane.

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators

“…In order to confirm the directionality of Cdr1p-, Cdr2p-and Cdr3p-associated phospholipid translocation, in the following experiments we used fluorescamine, a fluorescent dye, which we had previously used routinely (Balasubramanian and Gupta, 1996;Dogra et al, 1999) to label exposed PE in respective disruptant strains. Fluorescamine labelling revealed an enhanced amount of exposed PE in CDR3 disruptant cells as compared to its parent strain, CIB4 ( Figure 5A).…”

“…In one of the studies Cerbon and Calderon (1991) have shown that the surface potential could regulate in-to-out translocation of phospholipids. Recently it has been observed that the phospholipid translocation between the two monolayers could be protein mediated in yeast cells (Balasubramanian and Gupta, 1996;Tang et al, 1996;Kean et al, 1997;Dixit and Gupta, 1998). We had earlier shown that a drug extrusion pump CDR1 of C. albicans could mediate aminophospholipid translocation .…”

“…In Saccharomyces cerevisiae aminophospholipids are preferentially located in the cytoplasmic leaflet of the plasma membrane (Balasubramanian and Gupta, 1996;Kean et al, 1997;Dixit and Gupta, 1998). Several mechanisms that could maintain membrane lipid asymmetry in yeast cells have been suggested.…”

“…This approach has commonly been used to determine the mechanism of transbilayer movement of phospholipids in eukaryotic cells (Kean et al, 1997;Balasubramanian and Gupta, 1996;Bevers et al, 1999). When labelled cells expressing CDR1 or CDR2 were incubated with NEM, significant reduction was observed in the backexchange of M-C 6 -NBD-PE (30-50% inhibition) to acceptor vesicles.…”

Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

“…Azide has been used to study the energy dependence of lipid and protein transport (3,40). For example, inhibition of the classical secretory pathway by azide has been taken as an indication of the ATP dependence of vesicle-mediated protein translocation.…”