A Novel Membrane Protein, Ros3p, Is Required for Phospholipid Translocation across the Plasma Membrane inSaccharomyces cerevisiae

Abstract

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3⌬ cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergentinsoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.Phospholipids in most biological membranes are arranged asymmetrically between the two leaflets of the bilayer. In eukaryotic plasma membrane, aminophospholipids such as phosphatidylethanolamine (PE) 1 and phosphatidylserine (PS) reside predominantly in the inner leaflet, whereas phosphatidylcholine (PC) and sphingolipids are enriched in the outer leaflet (1-3). This transbilayer distribution of membrane lipids is not a static situation but is likely to be a result of the balance between the inward and outward translocation of phospholipids across the bilayer membranes (4). The rapid translocation of phospholipids between the outer and the inner leaflets of plasma membranes has been detected in various mammalian and yeast cells by fluorescence-labeled or short-chain analogs of phospholipids (5-7).In budding yeast, it is shown that the fluorescence-labeled analogs of phospholipids, including PC, PE, and PS, are translocated across the plasma membrane and that this translocation is not significantly prevented in the end and vps yeast mutants in which intracellular vesicular transport, including endocytosis, is impaired (8 -11). Because t...

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

“…lem3 and dnf1∆ dnf2∆ mutants, but not the dnf1∆ or dnf2∆ single mutant, are hypersensitive to cinnamycin [10,14], consistent with the defects in NBD-PE internalization in these mutants.…”

“…Unexpectedly, Lem3p(R316W) also coimmunoprecipitated with Dnf2p-13Myc, suggesting that the Lem3p(R316W)-Dnf2p complex is formed in the ER, but is not transported out of the ER due to the action of the ER qual-ity control mechanism. Kato et al showed that Lem3p/Ros3p was associated with the detergent-insoluble fraction [14]. As shown in Fig.…”

“…Mouse anti-HA (HA.11) and anti-Myc (9E10) monoclonal antibodies were purchased from Babco (Richmond, CA) and Sigma-Aldrich (St. Louis, MO), respectively. Rabbit anti-Lem3p polyclonal antibodies have been described previously [14]. The horseradish peroxidase-conjugated secondary antibodies (sheep anti-mouse IgG and donkey anti-rabbit IgG) used for immunoblotting were purchased from Amersham Biosciences (Piscataway, NJ).…”

“…The preparation of large unilamellar vesicles was performed as described previously [13]. Fluorescently labeled phospholipid internalization experiments were performed as described previously [13,14]. Briefly, cells carrying the designated plasmids were grown to early-mid log phase in SD-Leu media at 30°C or 37°C.…”

“…Cdc50p and its homolog, Lem3p, were subsequently shown to associate with Drs2p and Dnf1p, respectively, and to be required for the exit of these P-type ATPases from the endoplasmic reticulum (ER) [13]. Thus, disruption of the LEM3 gene results in a marked decrease in the internalization of fluorescencelabeled analogs of PE and PC across the plasma membrane [13][14][15]. The Lem3p-Dnf1p and Cdc50p-Drs2p complexes are functionally redundant, because simultaneous 4 loss of function of both complexes results in a synthetic growth defect.…”