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Mol Biol Cell. Nov 2004; 15(11): 4949–4959.
PMCID: PMC524749

Cross Talk between Sphingolipids and Glycerophospholipids in the Establishment of Plasma Membrane Asymmetry

Howard Riezman, Monitoring Editor


Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.


Lipid molecules are not equally distributed between the inner and outer leaflets of the lipid bilayer forming the plasma membrane. For example, phosphatidylcholine (PC) is abundant in the outer leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) are predominantly found in the inner leaflet (Schroit and Zwaal, 1991 blue right-pointing triangle). The trans-bilayer movement of phospholipids in a membrane model is very slow (Kornberg and McConnell, 1971 blue right-pointing triangle) in contrast to such movement through biogenic membranes. Thus, the existence of enzymes that catalyze the translocation of phospholipids has long been suspected. Recent studies have demonstrated that three major groups of enzymes, P-type ATPases, ATP-binding cassette (ABC) transporters, and scramblases, are active in the trans-bilayer movement of glycerophospholipids (Bevers et al., 1999 blue right-pointing triangle; Williamson and Schlegel, 2002 blue right-pointing triangle).

One particular subfamily of the P-type ATPases, the aminophospholipid translocases, are involved in the inward movement (flip) of PS and PE. Mammalian ATPase II, the product of the class 1a gene, was the first aminophospholipid translocase identified (Tang et al., 1996 blue right-pointing triangle). The yeast Saccharomyces cerevisiae contains five such translocases: Drs2p, Dnf1p, Dnf2p, Dnf3p, and Neo1p. Deletion of the DRS2 gene reportedly resulted in a loss in the uptake of labeled PS (Tang et al., 1996 blue right-pointing triangle), although contrary results have been presented by other groups (Siegmund et al., 1998 blue right-pointing triangle; Marx et al., 1999 blue right-pointing triangle). Recently, Dnf1p and Dnf2p were found to be involved in glycerophospholipid translocation in the plasma membrane (Pomorski et al., 2003 blue right-pointing triangle). That report demonstrated that Drs2p is not localized in the plasma membrane but rather is found in the Golgi complex where it functions in the flip of glycerophospholipids (Pomorski et al., 2003 blue right-pointing triangle). Dnf3p, another P-type ATPase closely related to the three proteins mentioned above, seems to have overlapping functions. Mutants carrying the quadruple deletion Δdrs2 Δdnf1 Δdnf2 Δdnf3 were inviable; however, triple mutants (Δdrs2 Δdnf1 Δdnf2, Δdrs2 Δdnf2 Δdnf3, Δdrs2 Δdnf1 Δdnf3, and Δdnf1 Δdnf2 Δdnf3) survived (Hua et al., 2002 blue right-pointing triangle). Single Δdrs2 mutants exhibited late Golgi defects such as decreased processing of pro-α-factor and an accumulation of abnormal Golgi cisternae (Chen et al., 1999 blue right-pointing triangle). Additionally, transport of alkaline phosphatase to the vacuole was defective in the Δdrs2 Δdnf1 cells (Hua et al., 2002 blue right-pointing triangle). Moreover, the Δdnf1 Δdnf2 Δdnf3 mutation caused a mislocalization of GFP-tagged Snc1p, suggesting the involvement of Dnf1p, Dnf2p, and Dnf3p in the endosome-to-trans-Golgi network transport (Hua et al., 2002 blue right-pointing triangle). Finally, a defect in endocytosis was observed in the Δdnf1 Δdnf2 Δdrs2 mutants (Pomorski et al., 2003 blue right-pointing triangle). These data suggest that proper glycerophospholipid asymmetry is important in the maintenance of organelle structure and intracellular trafficking.

An additional gene, ROS3/LEM3, was identified in separate studies screening mutants hypersensitive to a tetracyclic peptide that binds specifically to PE (Kato et al., 2002 blue right-pointing triangle) or resistant to alkylphosphocholine drugs (Hanson et al., 2003 blue right-pointing triangle). ROS3 is required for glycerophospholipid translocation across the plasma membrane (Kato et al., 2002 blue right-pointing triangle; Hanson et al., 2003 blue right-pointing triangle). Recently, Ros3p and its homolog Cdc50p were shown to interact with Dnf1p and Drs2p, respectively, and to function in the establishment of cell polarity (Saito et al., 2004 blue right-pointing triangle).

Certain ABC transporter family members are involved in the outward movement (flop) of glycerophospholipids. For instance, the transporter human MDR1 acts as a lipid translocase with broad specificity, whereas mouse mdr2 and human MDR3 function specifically in the translocation of PC (Smit et al., 1993 blue right-pointing triangle; Smith et al., 1994 blue right-pointing triangle; van Helvoort et al., 1996 blue right-pointing triangle). More recently, involvement in the outward transport of labeled PS was demonstrated for the ABC transporter MRP1 by using red blood cells (Dekkers et al., 1998 blue right-pointing triangle). In yeast, Pdr5p and Yor1p, also of the ABC transporter family, have been shown to be involved in the flop of PE (Decottignies et al., 1998 blue right-pointing triangle).

In addition to glycerophospholipids, sphingolipids are major components of the eukaryotic plasma membrane. Mammalian sphingolipids include sphingomyelin and hundreds of glycosphingolipids, whereas in yeast such as S. cerevisiae only three sphingolipids exist, all containing myo-inositol. Ceramide, the backbone of sphingolipids, is composed of a long-chain base (LCB) and a fatty acid linked by an amide bond. In mammalian cells the major LCB is sphingosine. Again, sphingosine does not exist in S. cerevisiae; instead, dihydrosphingosine (DHS) and phytosphingosine (PHS) serve as LCBs. In mammalian cells, sphingomyelin metabolites such as ceramide, sphingosine, and sphingosine 1-phosphate act as bioactive lipid molecules to regulate cell growth, differentiation, motility, and apoptosis (Hannun et al., 2001 blue right-pointing triangle; Spiegel and Milstien, 2003 blue right-pointing triangle). In yeast, the two LCBs and their phosphorylated forms, the long-chain base 1-phosphates (LCBPs), also function as signaling molecules involved in heat stress response, endocytosis, cell cycle arrest, Ca2+ mobilization, and diauxic shift (Obeid et al., 2002 blue right-pointing triangle).

Sphingomyelin and glycosphingolipids are known to be localized in the outer leaflet of the plasma membrane, whereas the distribution of ceramide and LCBs between the two leaflets has not been determined. Previous biochemical analysis by using mouse liver suggested that sphingolipid synthesis is initiated and also proceeds on the cytoplasmic side of the endoplasmic reticulum (ER) membrane, to the point of dihydroceramide synthesis (Mandon et al., 1992 blue right-pointing triangle). LCBs seem to be located on both sides of the ER membrane. LCBs are converted to LCBPs by LCB kinases localized in the cytoplasm and can associate peripherally with the membrane on the cytoplasmic side. However, the LCBP phosphatase Lcb3p also generates LCBs from LCBP in the ER lumen (Kihara et al., 2003 blue right-pointing triangle). Because galactosylceramide and glucosylceramide are synthesized on the luminal side of the ER and on the cytoplasmic side of the Golgi apparatus, respectively (Coste et al., 1986 blue right-pointing triangle; Futerman and Pagano, 1991 blue right-pointing triangle; Jeckel et al., 1992 blue right-pointing triangle; Sprong et al., 1998 blue right-pointing triangle), their precursor, ceramide, also may exist in both sides. Once synthesized, glucosylceramide on the cytoplasmic side is translocated to the luminal side of the Golgi apparatus, where conversion to lactosylceramide and complex glycosphingolipids takes place (Lannert et al., 1994 blue right-pointing triangle). Sphingomyelin is also synthesized in the luminal side of the Golgi apparatus (Futerman et al., 1990 blue right-pointing triangle; Jeckel et al., 1992 blue right-pointing triangle).

Although the mechanism for glycerophospholipid translocation has been well studied, a similar system for sphingolipid translocation has yet to be disclosed. Recently, Rsb1p was identified as a putative LCB transporter/translocase (Kihara and Igarashi, 2002 blue right-pointing triangle). Overexpression of Rsb1p resulted in an increased efflux of LCBs, whereas disruption of its gene greatly reduced such efflux (Kihara and Igarashi, 2002 blue right-pointing triangle). Here, we reveal that Rsb1p expression is enhanced by the deletion of the ABC transporter genes PDR5 and YOR1. Moreover, the expression was up-regulated in mutants defective in the inward translocation of glycerophospholipids. These results suggest that altered membrane asymmetry can trigger the expression of RSB1.


Yeast Strains, Plasmids, and Media

S. cerevisiae strains used are listed in Table 1. The Δdpl1::TRP1 (Kihara and Igarashi, 2002 blue right-pointing triangle), Δrsb1::HIS3 (Kihara and Igarashi, 2002 blue right-pointing triangle), and Δlcb4::LEU2 (Kihara et al., 2003 blue right-pointing triangle) constructions were described previously. The Δpdr5::KanMX4, Δdhh1::KanMX4, Δpdr1::KanMX4, Δyor1::KanMX4, Δros3::KanMX4, Δdnf1::KanMX4, Δdnf2::KanMX4, Δpdr3::KanMX4, Δros3::HIS3, and Δyor1::HIS3 cells were constructed by replacing their entire open reading frames with the KanMX4 marker or the HIS3 marker. For construction of the Δpdr5::URA3 and Δdnf1::HIS3 cells, the 2.0-kb BamHI-PmeI region in the PDR5 gene and the 0.5-kb SmaI-NruI region in the DNF1 gene were replaced with the respective auxotrophic markers. The cells were grown either in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or in synthetic complete (SC) medium (0.67% yeast nitrogen base and 2% glucose) containing nutritional supplements.

Table 1.
Yeast strains used in this study

The pAK80 plasmid is a yeast cloning vector for TDH3 promoter-dependent expression (Kihara and Igarashi, 2002 blue right-pointing triangle). The pAK90 and pAK170 plasmids are derivatives of pAK80 and encode RSB1 and 3xHA-RSB1, respectively (Kihara and Igarashi, 2002 blue right-pointing triangle).

Construction of KHY426 (rsb1::3xHA-RSB1) cells was as follows. KHY403 (SEY6210, Δrsb1::URA3) cells were first created by replacing the 0.6-kb EcoRV-EcoRV region in the RSB1 gene with the URA3 marker. The pAK464 plasmid, in which the 5′-upstream region (-317 to -1) of the RSB1 gene was connected to the 3xHA-RSB1 construct derived from the pAK170 plasmid, and the primers 5′-CCAGAATCAATAGAAATAAGAAGAG-3′ and 5′-TATGGAGGTAAACTAACGCTCCTGC-3′ were used for amplification of the 5′-upstream-3xHA-RSB1 region by polymerase chain reaction (PCR). Thus, generated fragments were then introduced into the KHY403 cells, and cells that lost the URA3 marker were selected with 50 μg/ml 5-fluoro-orotic acid. Genomic DNAs were prepared from some of the obtained colonies, and replacement of Δrsb1::URA3 with 3xHA-RSB1 was examined by PCR. One of the clones, KHY426, exhibited the rsb1::3xHA-RSB1 genotype.

To identify the 5′-upstream region of the RSB1 gene required for the expression of Rsb1p, pAK556, pAK549, and pAK550 were created as follows. Each 5′-upstream-3xHA-RSB1 region was amplified by PCR by using genomic DNA prepared from KHY426 as a template, a common primer (5′-GTACATATTACGATGTCGAAATATAAGG-3′), and respective primers (pAK556, 5′-TTAGAGCGCGTGTTGAAATATAGTC-3′; pAK549, 5′-AGCATTCTTGCTCCGTCATATTTCC; pAK550, 5′-AAGATATGGTCTCCGTCGGTCCTCTG-3′). The amplified region was then inserted into the yeast expression vector pRS423 (Christianson et al., 1992 blue right-pointing triangle).

Isolation of a PHS-resistant Mutant by Transposon Mutagenesis

A yeast genomic library that had been mutagenized by random insertion of the transposon mTn-lacZ/LEU2 (Burns et al., 1994 blue right-pointing triangle) was kindly provided by Dr. Michael Snyder (Yale University, New Haven, CT). The genomic library was digested with NotI to excise yeast DNA fragments, and the resulting fragments were used for the transformation of KHY13 cells. Transformants were selected by incubating on SC medium lacking leucine. Pooled transformants were then plated on YPD medium containing 15 μM PHS and 0.0015% Nonidet P-40 as a dispersant. After incubating at 30°C for 2 d, we obtained PHS-resistant mutants at a frequency of about 1 to 9000. The sites of insertion were then determined according to the manual of the Yale Genome Analysis Center (http://ygac.med.yale.edu/).

Assaying [3H]DHS Uptake and Release

Yeast cells grown at 30°C to 1 OD600 unit/ml were treated with [4,5-3H]DHS (0.5 μCi/1 OD600 cells) (50 Ci/mmol) (American Radiolabeled Chemicals, St. Louis, MO), which had been complexed with 1 mg/ml fatty acid-free bovine serum albumin (BSA) (A-6003; Sigma-Aldrich, St. Louis, MO), and incubated for various time periods. At each time point, cells equivalent to 0.45 OD600 were chilled on ice, washed twice with growth medium containing 1 mg/ml BSA, and suspended in 100 μl of water, for lipid extraction, or in 500 μl of medium containing 1 mg/ml BSA, for use in a DHS release assay. In the DHS release assay, cells were incubated at 30°C for 10 min, and cells and medium were separated by centrifugation. Cells were then suspended in 100 μl of water. Lipids were extracted from both cells and medium by successive addition and mixing of 3.75 volumes of chloroform/methanol/HCl [100:200:1 (vol:vol)], 1.25 volumes of chloroform, and 1.25 volumes of 1% KCl. Phases were separated by centrifugation, and the organic phase was recovered, dried, and suspended in chloroform/methanol [2:1 (v/v)]. The labeled lipids were resolved by thin layer chromatography (TLC) on Silica Gel 60 high-performance TLC plates (Merck, Whitestation, NJ) with 1-butanol/acetic acid/water [3:1:1 (v/v)].


Immunoblotting was performed as described previously (Kihara and Igarashi, 2002 blue right-pointing triangle) by using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ) or Lumi-LightPLUS Western blotting substrate (Roche Diagnostics, Mannheim, Germany). Anti-hemagglutinin (HA) (Y-11) antibody (0.2 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Dpm1 antibody (2 μg/ml; Molecular Probes, Eugene, OR), anti-Pma1 (yN-20) antibody (0.4 μg/ml; Santa Cruz Biotechnology), horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG F(ab′)2 fragment (1:7500 dilution; Amersham Biosciences), and HRP-conjugated donkey anti-goat IgG (0.08 μg/ml; Santa Cruz Biotechnology) were used.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was prepared using the YeaStar RNA kit (Zymo Research, Orange, CA) according to the manufacturer's manual. The RNA was converted to cDNA by using the primer RSB1-R (5′-AATCGGGTATTTCATGCTTGCTTGG-3′) and the ProSTAR first strand RT-PCR kit (Stratagene, La Jolla, CA). The RSB1 cDNA was then amplified by PCR by using primers RSB1-F (5′-TTGTCATTTGGGGTATACTACTGAC-3′) and RSB1-R. RT-PCR for ACT1 mRNA also was performed as a control by using the primer ACT1-R (5′-AACACTTGTGGTGAACGATAGATGG-3′) for preparing cDNA and primers ACT1-F (5′-GATTCTGGTATGTTCTAGCGCTTGC-3′) and ACT1-R for subsequent PCR.

Sucrose Gradient Fractionation

Cells grown in YPD medium were treated with 10 mM sodium azide. Approximately 4 × 108 cells were converted to spheroplast and lysed in 10% sucrose solution in buffer I (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1× protease inhibitor mixture [Complete; Roche Diagnostics, Indianapolis, IN], and 1 mM phenylmethylsulfonyl fluoride), by using an electric Potter homogenizer for 10 strokes. After removal of cell debris by centrifugation, total lysates (0.4 ml) were overlaid on a step sucrose gradient (0.4 ml of 55% sucrose, 1 ml of 45% sucrose, and 0.8 ml of 30% sucrose in buffer I) and centrifuged at 50,000 rpm (256,000 × g) in an S52ST rotor (Hitachi Koki, Tokyo, Japan) for 5 h. Fractions were collected from the top and separated by SDS-PAGE, followed by immunoblotting.

7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Lipid Trafficking Assay

Yeast cells were grown at 30°C in SC medium to 1 OD600 unit/ml, chilled on ice, and treated with 1/100 volume of 500 μM myristoyl-(NBD-hexanoyl)-PE (NBD-PE) (Avanti Polar Lipids, Alabaster, AL) or 500 μM myristoyl-(NBD-hexanoyl)-PC (NBD-PC) (Avanti Polar Lipids) while vigorously mixing. Cells were incubated at 4°C for 30 min and washed with cold SC medium. Cells assayed for flip were suspended in 100 μl water before lipid extraction. Cells being assayed for flop were further incubated in SC medium at 30°C for 0 or 30 min, washed with cold SC medium, and suspended in 100 μl of water.

Lipids were extracted by the successive addition and mixing of 375 μl chloroform/methanol [1:2 (v/v)], 125 μl of chloroform, and 125 μl of 1% KCl. Phases were separated by centrifugation, and the organic phase was recovered, dried, and suspended in chloroform. The lipids were resolved by TLC on Silica Gel 60 high-performance TLC plates (Merck) with chloroform/methanol/28% NH3/water [50:40:7:3 (v/v)]. NBD-lipids were quantified using a Bio Imaging analyzer FLA-2000 (Fuji Photo Film, Kanagawa, Japan).


Isolation of PHS-resistant, Transposon-inserted Mutants

Dpl1p is a LCBP lyase that degrades phytosphingosine 1-phosphate (PHS1P) to fatty aldehyde and phosphoethanolamine. In yeast cells, PHS is imported from the medium and converted into PHS1P by the LCB kinase Lcb4p. Over-accumulation of PHS1P is toxic to cells (Kim et al., 2000 blue right-pointing triangle; Zhang et al., 2001 blue right-pointing triangle), and Δdpl1 mutants are more sensitive to exogenous PHS than wild-type cells (Saba et al., 1997 blue right-pointing triangle). Using this phenotype, we previously screened for a multicopy suppressor of the PHS-sensitive phenotype of the Δdpl1 mutants and obtained the RSB1 gene (Kihara and Igarashi, 2002 blue right-pointing triangle). We further demonstrated that this gene product is a putative LCB-transporter/translocase. In the present study, we searched for additional LCB/LCBP-related genes by using another approach, transposon mutagenesis.

The genomes of KHY13 (Δdpl1) cells were randomly mutated by insertion of the mTn-lacZ/LEU2 transposon. Pooled mutants were then plated on YPD medium, containing 15 μM PHS, and incubated at 30°C for 2 d. Several clones were obtained at a frequency of about 1 to 9000 mutants. Of these, 26 mutants were subjected to further analyses. To identify genes disrupted by the transposon, yeast genomic DNA adjacent to the transposon was cloned from each mutant. Sequence analyses revealed four classes of mutants, based on the genes interrupted by the transposon. Eighteen mutants contained the transposon insertion within the LCB4 gene. Isolation of lcb4 mutants had been expected, because Lcb4p is a major LCBP-synthesizing enzyme. Five mutants carried insertions in PDR5, the gene that encodes the ABC transporter Pdr5p, which reportedly confers resistance to a wide range of compounds (Rogers et al., 2001 blue right-pointing triangle). Two others were mutants of the DHH1 gene, which encodes a putative RNA helicase that acts as a component of a CCR4-NOT transcriptional regulatory factor complex (Hata et al., 1998 blue right-pointing triangle; Maillet and Collart, 2002 blue right-pointing triangle). One mutation was inserted by the transposon in the YRM1 gene, normally encoding a transcription factor (Lucau-Danila et al., 2003 blue right-pointing triangle).

Among the isolated mutants, the lcb4 mutants exhibited the strongest phenotype with regard to PHS resistance. Control KHY360 (Δdpl1) cells (KHY13 cells carrying a wild-type LEU2 gene to control for the LEU2 transposon) were not able to grow in the presence of 15 μM PHS, yet an lcb4 mutant, KHY288, was not similarly inhibited (Table 2). Additionally, the pdr5 mutants were highly resistant to PHS. Although their growth rate in 15 μM PHS was slightly reduced, that at 12.5 μM was not affected (Table 2). In contrast, the PHS resistance of the dhh1 mutants was rather weak, because their growth in 15 μM PHS was obviously retarded (Table 2). The yrm1 mutants, KHY294, were the most sensitive to PHS of the obtained mutants, with only a slight difference in the sensitivity between them and the KHY360 cells (Table 2).

Table 2.
PHS sensitivity of the isolated mutants

We next constructed deletion mutants for LCB4, PDR5, DHH1, or YRM1 in the Δdpl1 background and investigated their PHS sensitivity. KHY22 (Δdpl1 Δlcb4), KHY364 (Δdpl1 Δpdr5), and KHY365 (Δdpl1 Δdhh1) cells exhibited PHS resistance identical to that of their respective transposon mutants (Table 2), indicating that their gene disruptions were indeed responsible for the PHS resistance. However, the Δdpl1 Δyrm1 mutants did not show any PHS resistance (our unpublished data). For further analyses we used the pdr5 mutants, because these exhibited the most striking PHS sensitivity, excluding the already characterized lcb4p.

Efflux of LCB Is Enhanced in pdr5 Mutants

We first investigated whether the pdr5 mutation affects the intracellular accumulation of LCBs. KHY13 (PDR5+), KHY299 [pdr5 (pdr5::mTn-lacZ/LEU2)], and KHY323 (Δpdr5) cells were labeled with [3H]DHS for 5, 20, and 60 min at 30°C. Then, intracellularly accumulated DHS was extracted, separated by TLC, and quantified. As shown in Figure 1, wild-type cells accumulated DHS in a time-dependent manner, so that ~40% of the added DHS had accumulated intracellularly by 60 min. On the other hand, accumulation of [3H]DHS in the Δpdr5 mutants was reduced, to ~70% of that in wild-type cells (Figure 1). Likewise, the transposoninserted mutant KHY299 exhibited a decrease in the DHS accumulation similar to that of the pdr5 deletion mutants (Figure 1).

Figure 1.
DHS accumulation decreases in pdr5 mutants. KHY13 (PDR5+; circles), KHY299 (pdr5::mTn-lacZ/LEU2; triangles), and KHY323 (Δpdr5; squares) cells grown in YPD medium were labeled with [3H]DHS complexed with BSA, for 5, 20, and 60 min. Cellular lipids ...

Reduced accumulation of DHS could be caused by a decrease in its uptake or an increase in its efflux. To examine the second possibility, we next performed a DHS release assay. Wild-type and Δpdr5 cells were labeled with [3H]DHS for 1 h at 30°C to allow cells to accumulate DHS. Cells were then washed and incubated for 10 min at 30°C. Lipids were extracted from both media and cells and separated by TLC. Wild-type cells released 7% of the accumulated DHS, whereas the Δpdr5 cells exported 22% (Figure 2), indicating increased DHS release.

Figure 2.
DHS release is increased in pdr5 mutants in an RSB1-dependent manner. (A) KHY400 (wild-type; lane 1), KHY452 (pdr5; lane 2), KHY425 (pdr5 Δyor1; lane 3), KHY427 (Δrsb1; lane 4), KHY454 (pdr5 Δrsb1; lane 5), and KHY424 (pdr5 Δ ...

RSB1 Is Involved in Enhanced LCB Export Caused by the pdr5 Mutation

The possibility that Pdr5p normally acts as an efflux pump for DHS was deemed unlikely, because the DHS release would have been reduced, not increased, by the pdr5 mutation. Instead, the effect seemed to be indirect. Rsb1p would be a likely candidate for a protein influenced by a pdr5 mutation, because we recently identified it as a putative transporter/translocase for LCBs including PHS, DHS, and sphingosine (Kihara and Igarashi, 2002 blue right-pointing triangle). To investigate this hypothesis, we created Δdpl1 Δrsb1 mutants and Δdpl1 pdr5 Δrsb1 mutants. KHY427 cells (Δdpl1 Δrsb1) were slightly more sensitive to PHS than KHY400 cells (Δdpl1) (Table 3), due to a reduction in the efflux of PHS (Kihara and Igarashi, 2002 blue right-pointing triangle; Figure 2). Although KHY452 cells (Δdpl1 pdr5) were resistant to PHS, as described above, introducing the Δrsb1 mutation caused the resulting cells (KHY454) to become as sensitive to PHS as the KHY427 cells (Table 3). This indicated that the PHS-resistant phenotype of the pdr5 mutants was completely dependent on RSB1. Furthermore, the [3H]DHS release assay demonstrated that the efflux, unequivocally exhibited by wild-type cells (7.5%), was greatly reduced in the Δrsb1 mutant (0.9%) (Figure 2). Thus, Rsb1p is responsible for most of the DHS export activity in wild-type cells. Consistent with their PHS sensitivity, the pdr5 Δrsb1 cells export only slight amounts of DHS (1.3%) that are almost identical to those released by the Δrsb1 mutant (Figure 2).

Table 3.
The PHS-resistant phenotype of the pdr5 mutant is dependent on RSB1

We next searched for other ABC transporter genes involved in the PHS resistance, together with the PDR5 gene. For this purpose, we introduced several deletion mutations into KHY452 (Δdpl1 pdr5) cells and investigated their PHS sensitivity. These mutations included Δpdr10, Δpdr12, Δpdr15, Δpdr18, Δsnq2, and Δyor1. Of these, only KHY425 (Δdpl1 pdr5 Δyor1) cells exhibited enhanced PHS resistance, compared with the KHY452 cells (Table 3; our unpublished data). However, Δdpl1 Δyor1 mutants were not PHS resistant (our unpublished data), indicating that the Δyor1 mutation was effective only when combined with the pdr5 mutation. Additionally, the DHS release assay demonstrated that the pdr5 Δyor1 mutants exported approximately twofold more DHS than the pdr5 mutants did (Figure 2), confirming that the enhanced PHS resistance caused by the Δyor1 mutation was due to an increased efflux of PHS. The PHS-resistant phenotype of the Δdpl1 pdr5 Δyor1 cells was again RSB1 dependent, because KHY424 (Δdpl1 pdr5 Δyor1 Δrsb1) cells were sensitive to PHS (Table 3), and the export rate of DHS by the pdr5 Δyor1 Δrsb1 mutants was very low (Figure 2).

Up-Regulation of Rsb1p in Δpdr5 and Δpdr5 Δyor1 Mutants

We next considered whether the enzyme activity or the amount of Rsb1p was increased in the pdr5 and pdr5 Δyor1 mutants. To examine the enzyme activity of Rsb1p, we introduced the pAK170 plasmid, encoding N-terminally, triple HA (3xHA)-tagged RSB1 (3xHA-RSB1), into both the KHY458 (Δrsb1) and KHY424 (Δrsb1 pdr5 Δyor1) cells. Because this 3xHA-RSB1 fusion gene was designed to be expressed constitutively under the control of the TDH3 promoter, the amounts of Rsb1p were expected to be constant among the cells. Indeed, immunoblotting by using an anti-HA antibody demonstrated that KHY458 cells bearing pAK170 expressed 3xHA-Rsb1p at levels nearly equal to those of KHY424 cells bearing pAK170 (Figure 3A). Introduction of the pAK170 plasmid into the KHY458 cells resulted in an ~12-fold increase in DHS release; a similar increase also was observed in the KHY424 cells bearing pAK170 (Figure 3B). These results suggested that the activity per Rsb1p molecule was not affected in the pdr5 Δyor1 cells. We also looked for differences in the intracellular localization of Rsb1p between the pdr5 and pdr5 Δyor1 mutants, but we found that the sucrose gradient fractionation profiles of 3xHA-Rsb1p expressed in wild-type and in the pdr5 Δyor1 cells were similar (our unpublished data).

Figure 3.
The enzyme activity of Rsb1p is unchanged in pdr5 Δyor1 cells. (A) KHY458 cells (Δrsb1) bearing pAK80 (vector) (lane 1) or pAK170 (3xHA-RSB1) (lane 2) and KHY424 cells (Δrsb1 pdr5 Δyor1) bearing pAK80 (lane 3) or pAK170 ...

Therefore, we next examined the possibility that the amount of Rsb1p was increased in the pdr5 and pdr5 Δyor1 cells. To detect endogenous Rsb1p, the RSB1 gene was replaced with 3xHA-RSB1, with the RSB1 promoter remaining intact, creating KHY426 cells. KHY444 (Δpdr5), KHY515 (Δyor1), and KHY533 (Δpdr5 Δyor1) cells were then produced by introducing the Δpdr5 and/or Δyor1 mutations into the KHY426 cells. Immunoblotting by using an anti-HA antibody demonstrated that 3xHA-Rsb1p was increased in the Δpdr5 cells compared with the control KHY426 cells (Figure 4A). Although the Δyor1 single mutation did not affect the amount of 3xHA-Rsb1p, the Δpdr5 Δyor1 double mutation further enhanced the expression of 3xHA-Rsb1p compared with the Δpdr5 single mutation (Figure 4A). These protein amounts correlated well with the PHS sensitivity (Table 3) and the DHS export activity of the respective mutations (Figure 2).

Figure 4.
Expression of RSB1 is increased in Δpdr5 and Δpdr5 Δyor1 cells. (A) KHY426 (wild-type; lane 1), KHY444 (Δpdr5; lane 2), KHY515 (Δyor1; lane 3), and KHY533 (Δpdr5 Δyor1; lane 4) cells were grown in ...

To investigate whether the increases in Rsb1p amounts in the pdr5 and pdr5 Δyor1 cells resulted from changes at the transcriptional level, we performed an RT-PCR analysis. Total RNAs were isolated from KHY400 (wild-type), KHY452 (pdr5), and KHY425 (pdr5 Δyor1) cells and subjected to RT-PCR by using primers specific for RSB1, and for ACT1, encoding actin, as a control. As shown in Figure 4B, the amount of RSB1 amplified from RNA prepared from the pdr5 mutants was increased compared with that from wild-type cells. Moreover, the pdr5 Δyor1 double mutations exhibited an even higher amount (Figure 4B). These results clearly showed that Rsb1p is up-regulated in the pdr5 and pdr5 Δyor1 mutants at a transcriptional level.

In a previous study, we investigated the intracellular localization of 3xHA-Rsb1p by using immunofluorescence microscopy and found that 3xHA-Rsb1p is localized at both the ER and the plasma membrane (Kihara and Igarashi, 2002 blue right-pointing triangle). However, in that study, 3xHA-Rsb1p was overproduced under the control of the TDH3 promoter, and its distribution between the ER and the plasma membrane was not quantitatively assessed. Therefore, we investigated the localization in KHY533 cells expressing 3xHA-Rsb1p under its own promoter, by using sucrose gradient fractionation. As shown in Figure 4C, 3xHA-Rsb1p exhibited two peaks: a major peak in fraction 3 and a minor peak in fraction 6. The ER membrane protein Dpm1p and the plasma membrane protein Pma1p were highest in fractions 3 and 6, respectively. These results indicated that 3xHA-Rsb1p resides both at the ER and the plasma membrane, with the majority at the ER.

Expression of RSB1 Is Under the Control of Pdr1p

Pdr1p and Pdr3p are zinc finger-type transcription factors. Their gain-of-function mutations, such as the PDR1-3 and PDR3-7 mutations, cause cells to acquire a pleiotropic drug-resistant phenotype through the activation of several ABC transporter family members (Balzi et al., 1994 blue right-pointing triangle; Carvajal et al., 1997 blue right-pointing triangle; Nourani et al., 1997 blue right-pointing triangle; DeRisi et al., 2000 blue right-pointing triangle). Previous comprehensive microarray analysis investigating genes up-regulated by the PDR1-3 and/or PDR3-7 mutation revealed that the expression of RSB1 was enhanced, markedly by the PDR1-3 mutation and slightly by the PDR3-7 mutation (DeRisi et al., 2000 blue right-pointing triangle). Therefore, we investigated roles for Pdr1p and Pdr3p in the transcriptional regulation of the RSB1 gene, by using their disruptants. When the Δpdr1 mutation was introduced into KHY426 (3xHA-RSB1) cells, no expression of 3xHA-Rsb1p was detected in the lysate of the resulting strains (KHY469) by immunoblotting (Figure 5A). The increased expression of Rsb1p observed in the Δpdr5 and Δpdr5 Δyor1 cells also was diminished by the introduction of the Δpdr1 mutation (Figure 5A), indicating that the expression of Rsb1p was under the control of Pdr1p. In contrast, the Δpdr3 mutation had no effect on the Rsb1p expression in either background tested (PDR5+ YOR1+, pdr5 YOR1+, or pdr5 Δyor1) (Figure 5B).

Figure 5.
Transcription of the RSB1 gene is under the control of Pdr1p but not Pdr3p. Cells were grown in YPD medium at 30°C, and proteins were prepared from cell lysates. Total proteins (25 μg) were separated by SDS-PAGE, followed by detection ...

There are two TCCGCGGA nucleotide sequences in the RSB1 gene that match the pleiotropic drug response elements (PDREs; TCCG/AC/TGG/CA/G), situated 816 base pairs and 758 base pairs upstream of the translational initiation codon. We designated these sequences PDRE-1 (-816 to -809 from the start of the RSB1 gene) and PDRE-2 (-758 to -751). Because the PDREs are known to be recognized by Pdr1p (Katzmann et al., 1996 blue right-pointing triangle), we investigated whether PDRE-1 and PDRE-2 were required for the influence of Pdr1p on the expression of RSB1. For this purpose, we constructed three plasmids (pAK556, pAK549, and pAK550) of different lengths, carrying nucleotides found 5′-upstream of the RSB1 gene fused to the 3xHA-RSB1 construct, as illustrated in Figure 6A. 3xHA-Rsb1p was detected when pAK556, which contained both PDRE-1 and PDRE-2, was introduced into wild-type cells (Figure 6B, lane 1). However, its expression was not detected in KHY396 cells (Δpdr1) bearing pAK556 (Figure 6B, lane 4). In contrast, a marked increase in the amount of 3xHA-Rsb1p was observed in KHY421 cells (pdr5 Δyor1) harboring pAK556 (Figure 6B, lane 7). Expression of 3xHA-Rsb1p from pAK549, which carries only PDRE-2, was decreased both in wild-type and pdr5 Δyor1 cells, compared with that from pAK556 (Figure 6B). Moreover, pAK550, which lacks both PDRE-1 and PDRE-2, could not drive the 3xHA-Rsb1p expression in either cell type (Figure 6B). Thus, both PDRE-1 and PDRE-2 are required for full expression of RSB1.

Figure 6.
Both PDREs are required for expression of the RSB1 gene. (A) Schematic representation of the positions of PDRE-1 and PDRE-2 and the relative lengths of the constructed plasmids. (B) The plasmids pAK556 (lanes 1, 4, and 7), pAK549 (lanes 2, 5, and 8), ...

Expression of Rsb1p Is Increased in Δros3 and Δdnf1Δdnf2 Cells

Pdr5p and Yor1p are not only involved in drug efflux but also in translocation of PE (Decottignies et al., 1998 blue right-pointing triangle). High levels of Rsb1p were observed in Δpdr5 and Δpdr5 Δyor1 cells. However, because this occurred in the absence of drug treatment, we assumed that altered glycerophospholipid asymmetry was the cause of the high expression. Glycerophospholipid asymmetry is maintained by both P-type ATPase-dependent inward movement (flip) and by ABC transporter-dependent outward movement (flop). Therefore, we next examined whether the expression of Rsb1p would be increased by a reduced inward movement of glycerophospholipids. For this purpose, we disrupted two P-type ATPase-encoding genes, DNF1 and DNF2, in the 3xHA-RSB1 cells. Although neither a Δdnf1 nor Δdnf2 single mutation caused an increase in the amount of 3xHA-Rsb1p in either the PDR5+ or Δpdr5 background, Δdnf1 Δdnf2 double mutants exhibited a marked accumulation of 3xHA-Rsb1p (Figure 7A). A greater increase was observed in the Δpdr5 Δdnf1 Δdnf2 mutants (Figure 7A). The recently identified ROS3 gene is involved in the flip of glycerophospholipid (Kato et al., 2002 blue right-pointing triangle; Hanson et al., 2003 blue right-pointing triangle). As shown in Figure 7A, the amount of 3xHA-Rsb1p also was increased in Δros3 mutants and further elevated in Δros3 Δpdr5 mutants.

Figure 7.
The amounts of Rsb1p are increased in Δros3 and Δdnf1 Δdnf2 cells. (A) KHY426 (wild-type; lane 1), KHY444 (Δpdr5; lane 2), KHY531 (Δros3; lane 3), KHY532 (Δpdr5 Δros3; lane 4), KHY579 (Δ ...

The [3H]DHS release assay demonstrated that the amount of exported DHS was increased in both the Δros3 and Δdnf1 Δdnf2 mutants, compared with the wild-type cells (Figure 7B). Moreover, a further increase in the DHS release was observed in the pdr5 Δros3 and pdr5 Δdnf1 Δdnf2 cells (Figure 7B). Thus, the intracellular levels of Rsb1p correlated well with the amounts of released DHS.

Overproduction of Rsb1p Affects the Glycerophospholipid Translocation

The results mentioned above suggested that altered glycerophospholipid asymmetry modulates the distribution of LCBs between the two leaflets of the lipid bilayer by increasing the expression of Rsb1p. We investigated the possibility that altered LCB asymmetry resulting from changes in the Rsb1p levels affects the trans-bilayer movement of glycerophospholipids, by using the recently established flip-flop assay (Kean et al., 1997 blue right-pointing triangle; Hanson and Nichols, 2001 blue right-pointing triangle). Wild-type cells bearing the vector plasmid (pAK80) or those bearing the pAK90 plasmid that overproduces Rsb1p were incubated with NBD-PE or NBD-PC at 4°C for 30 min, allowing only the flip but not the flop reaction. For the flop assay, cells loaded with either lipid were further incubated at 30°C for 30 min. As shown in Figure 8A, cells overproducing Rsb1p flipped more NBD-PE (1.5-fold) and NBD-PC (2.6-fold) than control cells. On the other hand, flopping rates for both lipids were slightly reduced in cells overexpressing Rsb1p compared with cells bearing the vector plasmid (Figure 8B). These differences in the NBD-lipid flip/flop rates were not due to different sensitivities in these cells. Cells treated either with DMSO, 5 μM NBD-PE, or 5 μM NBD-PC at 4°C for 30 min were washed with medium, diluted, and incubated on SD-URA plates at 30°C. There were no differences in colony number or colony size. Indeed, the NBD-lipids exhibited no toxicity, at least at the concentrations used (our unpublished data). Thus, altered LCB asymmetry caused by overproduced Rsb1p affected the glycerophospholipid translocation, supporting the contention that alternations in the lipid asymmetry of glycerophospholipids and LCBs modulate the lipid distribution in a mutual manner.

Figure 8.
Overproduction of Rsb1p affects the flip-flop of NBD-labeled glycerophospholipids. KHY13 cells bearing pAK80 (vector; open columns) or pAK90 (RSB1; closed columns) plasmids were grown in SC medium lacking uracil at 30°C. (A) The cells were chilled ...


Glycerophospholipids in the plasma membrane are arranged asymmetrically between the two leaflets of the lipid bilayer. In erythrocytes, the best characterized system, PS, PE, and PI, are abundant in the inner leaflet, whereas PC is mainly located in the outer leaflet (Schroit and Zwaal, 1991 blue right-pointing triangle). Recent studies have revealed that certain P-type ATPases and ABC transporters are involved in their inward and outward movements, respectively (Bevers et al., 1999 blue right-pointing triangle; Williamson and Schlegel, 2002 blue right-pointing triangle). On the other hand, reports concerning factors regulating the topology of sphingolipids are very limited. We previously identified the RSB1 gene, which suppresses the LCB-sensitive phenotype of the Δdpl1 mutants when introduced as a multicopy plasmid (Kihara and Igarashi, 2002 blue right-pointing triangle). Subsequent studies suggested that Rsb1p is an ATP-dependent LCB transporter or translocase (Kihara and Igarashi, 2002 blue right-pointing triangle). Because LCBs are extremely hydrophobic and do not dissolve in aqueous solutions, LCBs added to culture medium may be incorporated spontaneously into the outer leaflet of the plasma membrane then be translocated to the inner leaflet by an unknown translocase. It is not clear whether Rsb1p is a transporter that pumps LCBs from the inner leaflet of the plasma membrane directly to the external medium, or a translocase (floppase) that flops LCBs from the inner leaflet to the outer leaflet. However, even if Rsb1p functions as a transporter, the released LCBs are likely then reincorporated into the plasma membrane, as shown previously (Kihara and Igarashi, 2002 blue right-pointing triangle); thus, their population in the outer leaflet is increased as well. So, whether Rsb1p is a transporter or a translocase, increases in Rsb1p would likely lead to the same result: changes in the asymmetry of LCB in the plasma membrane. In the medium used for the [3H]DHS release assay we included BSA, which has a high affinity for lipids. In the absence of BSA, the released DHS measured in the media was greatly reduced (our unpublished data). If Rsb1p is a translocase, LCBs flopped by Rsb1p toward the outer leaflet of the plasma membrane might be extracted by the exogenous BSA.

We favor the scenario that Rsb1p is a translocase rather than a transporter, because the majority of Rsb1p is localized at the ER (Figure 4C). This localization is reasonable, because its substrate LCBs are synthesized at the ER. In physiological conditions, Rsb1p may function to flop LCBs at the ER. Because lipids synthesized at the ER are transported to the plasma membrane via the Golgi apparatus by vesicular transport, without the loss of their membrane topology, the localization of the LCBs in the outer leaflet of the plasma membrane is likely established already at the ER. Therefore, in the [3H]DHS release assay, it is possible that the imported DHS is first transported to the ER, where it is translocated to the extracytoplasmic leaflet by ER-resident Rsb1p and then delivered to the plasma membrane. The other possibility is that the plasma membrane-localized Rsb1p flops the imported DHS to the outer leaflet of the plasma membrane. Indeed, LCBs are rapidly transported from the plasma membrane to the ER by either of two pathways. In the first pathway, LCBs are converted to LCBPs by the LCB kinase Lcb4p and then reconverted to LCBs at the ER by the LCBP phosphatase Lcb3p (Qie et al., 1997 blue right-pointing triangle; Mao et al., 1999 blue right-pointing triangle; Zanolari et al., 2000 blue right-pointing triangle; Funato et al., 2003 blue right-pointing triangle). In the second pathway, LCBs are directly transferred to the ER without conversion to LCBPs (Funato et al., 2003 blue right-pointing triangle). Although previous studies have shown that the first pathway is the major pathway (Qie et al., 1997 blue right-pointing triangle; Mao et al., 1999 blue right-pointing triangle; Zanolari et al., 2000 blue right-pointing triangle), the contribution of the second pathway is important in the yeast back-grounds used here, because exogenously added [3H]DHS is effectively metabolized at the ER in the Δlcb4 cells (our unpublished data).

Although overexpression of Rsb1p affected the trans-bi-layer movement of glycerophospholipids (Figure 7), deletion of the RSB1 gene had no effect (our unpublished data). Because the expression level of Rsb1p is very low in wild-type cells (Figure 4A), its activity also may remain low, at least under the growth conditions used here, which may explain the lack of any effect from the deletion. We speculate that in certain conditions affecting the glycerophospholipid asymmetry, Rsb1p is induced and begins to function.

In this study, we demonstrated that the expression of Rsb1p is increased in cells with altered glycerophospholipid asymmetry due to the disruption of either the inward or outward movement of glycerophospholipids (Figures (Figures44 and and7).7). Moreover, overexpression of Rsb1p affected both the inward and outward movements of glycerophospholipids (Figure 8). Thus, there may be a mechanism that maintains proper lipid asymmetry by regulating the trans-bilayer movement of both glycerophospholipids and sphingolipids. A mutant (end8) of LCB1, which normally encodes a subunit of a serine palmitoyltransferase involved in the first step of sphingolipid synthesis, was isolated and found to exhibit endocytosis defects (Zanolari et al., 2000 blue right-pointing triangle). LCBs, it was found, are required for endocytosis (Zanolari et al., 2000 blue right-pointing triangle). Certain P-type ATPase mutants also exhibit endocytosis defects (Pomorski et al., 2003 blue right-pointing triangle). Thus, glycerophospholipids and LCBs seem to function together in some cellular events occurring at the plasma membrane. We speculate that cells with improper glycerophospholipid asymmetry alter the membrane distribution of LCBs between the inner and outer leaflets by increasing the expression of Rsb1p, to compensate for certain decreased plasma membrane functions such as endocytosis and cell polarity.

This study revealed the existence of cross talk between glycerophospholipids and sphingolipids in establishing membrane asymmetry. Pdr1p seems to play an important role in this cross talk, because not only Rsb1p but also Pdr5p and Yor1p are under its control (Meyers et al., 1992 blue right-pointing triangle; Katzmann et al., 1995 blue right-pointing triangle). However, how signals of altered membrane asymmetry affect Pdr1p is largely unknown. As an intriguing model, we propose that an unidentified sensor protein recognizing the glycerophospholipid asymmetry transduces the signal to Pdr1p. Although Rsb1p is regulated at the transcriptional level, RT-PCR experiments demonstrated that the altered trans-bilayer movement of glycerophospholipids caused by the overproduction of Rsb1p was not due to changes in the mRNA levels of DNF1, DNF2, PDR5, or YOR1 (our unpublished data). Thus, the protein stability, activity, or intracellular localization of these enzymes may be affected by the change in the LCB asymmetry. However, further studies are required to reveal the precise molecular mechanism.


We are grateful to Dr. M. Snyder for providing mTn-lacZ/LEU2-mutagenized yeast genomic library. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (B) (12140201) and a Grant-in-Aid for Young Scientists (B) (15770078) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–06–0458. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–06–0458.


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