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J. Biol. Chem., Vol. 277, Issue 40, 37855-37862, October 4, 2002
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From the
Received for publication, June 5, 2002, and in revised form, July 11, 2002
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 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 the translocation reaction requires ATP and
is sensitive to sulfhydryl-modifying reagents (8, 9), the movements of
fluorescence-labeled phospholipids are likely to be facilitated by
proteins. Several molecules have been suggested to mediate the
transbilayer movements of phospholipids in yeast cells. Some members of
yeast ABC (ATP-binding cassette) transporters, such as Ste6p (12),
Pdr5p, and Yor1p (13), are shown to facilitate the
ATP-dependent outward movement of phospholipid analogs as well as amphipathic drugs. For the inward-directed translocation, an
integral membrane P-type ATPase, called Drs2p, was proposed to function
as an aminophospholipid translocase, because disruption of yeast
DRS2 gene resulted in a significant decrease of the
internalization of fluorescence-labeled PS across the plasma membrane
(14). However, several groups have failed to detect a difference
between wild type and drs2 Ro is a 19-amino acid tetracyclic polypeptide that strictly recognizes
the structure of PE and forms a tight equimolar complex with PE in
biological membranes (16, 17). The Ro peptide has become a useful tool
in monitoring the transbilayer movement of PE in biological membranes
(18, 19) and in studying the functional role of PE in cytokinesis (20,
21) and membrane protein folding (22). Because Ro peptide specifically
binds the cell surface PE and subsequently induces cytolysis (18), the
peptide is also useful for isolation of mutants with defective PE
synthesis as variants that are resistant to the cytolytic activity of
the peptide. We have previously isolated a peptide-resistant CHO-K1
cell mutant with specific decrease in cellular PE content, and we have
shown that the mutant is defective in intramitochondrial transport of phosphatidylserine (23).
In this study, we have isolated a yeast mutant, designated as
ros3, that shows hypersensitivity to the Ro peptide. We
found that the ros3 mutation was caused by defective
expression of the ROS3 gene product, Ros3p, an
evolutionarily conserved protein with two putative transmembrane
domains. We have performed a detailed analysis of the structure,
function, and subcellular localization of Ros3p, and we have shown that
Ros3p is a unique transmembrane protein present in lipid rafts.
Evidence that Ros3p plays an important role in regulating phospholipid
translocation across the bilayer membranes is presented in this paper.
Yeast Strains and Culture--
Saccharomyces
cerevisiae strain MHY501 (MAT Isolation of Ro-sensitive (ros) Mutant--
Mutagenesis with
ethyl methanesulfonate was carried out as described previously (25).
Mutagenized cells were plated for single colonies at 23 °C. Colonies
were replicated onto YPD plates containing 10 µM Ro
peptide and cultivated for 3 days at 30 °C. The negative colonies on
the Ro-containing plate were isolated from the master YPD plates.
Drug Sensitivity Assays--
For assays with lipid-binding
toxins, mid-log phase cultures were diluted to 1 × 106 cells/ml in YPD containing Ro peptide or amphotericin B
(Sigma) at indicated concentrations. After 24 h incubation at
30 °C, sensitivity was determined by measuring
A600 of cultures. Sensitivity to other agents such as SDS, Triton X-100, and ethanol was also determined as
described above.
Phospholipid Composition Analysis--
Mid-log phase cultures in
YPD were washed and resuspended in phosphate-buffered saline.
Phospholipids were extracted from the harvested cells (2-3 × 109 cells) and separated by two-dimensional thin layer
chromatography on silica plates. Solvent systems used for
chromatography are as follows: first dimension,
chloroform/methanol/acetic acid, 65:25:10 (v/v); and second dimension,
chloroform/methanol/formic acid, 65:25:10 (v/v). The amounts of
phospholipids were determined as described previously (26).
Cloning of the ROS3 Gene--
The ROS3 gene was
identified by its ability to complement the sensitivity to Ro peptide
of the ros3 mutant. The ros3 mutant was
transformed with the yeast genomic library, YCp50, which contains the
selection marker URA3 and the CEN element.
Candidate ROS3 clones were identified based on their ability
to recover growth in the presence of 10 µM Ro peptide.
Only one plasmid was isolated, and it was designated pS3. The pS3
contained an ~6.4-kb insert that was a portion of chromosome XIV
spanning the upstream of the ORF YNL323W through approximately
two-thirds of the ORF YNL320W. There are three full-length ORFs within
this region, YNL323W (LEM3), YNL322W (KRE1), and
an uncharacterized ORF YNL321W. To identify the complementary region of
the ros3 mutant, the 6.4-kb insert was cut into two
fragments. One was the 2.2-kb fragment containing the full-length
LEM3 gene and half of the KRE1 gene by digestion with SalI and KpnI. Another was the 4.2-kb
fragment containing the other half of the KRE1 gene, the
full-length YNL321W, and a portion of the ORF YNL320W by digestion with
KpnI and HindIII. The pS3-I carries the
SalI/KpnI fragment from pS3 in the pRS416 (URA3 and CEN) vector. The pS3-II carries the
KpnI/HindIII fragment from pS3 in the pRS416
vector. These plasmids were transformed into the ros3
mutant, and the ability to restore the growth of the ros3
mutant on the plates containing 10 µM Ro peptide was examined.
Production of Anti-Ros3p Antibody--
Polyclonal antibodies
against Ros3p were raised in New Zealand White female rabbits against
the synthetic peptides corresponding to the amino-terminal sequence
MVNFDLGQVGEVFRRKDKGC, according to the method described previously
(27). Antibodies were isolated from the immune sera of the rabbits by
affinity chromatography on a synthetic peptide-conjugated SulfoLink
column (Pierce).
Deletion of the ROS3 Gene--
The
SalI/KpnI fragment from pS3 was subcloned into a
pUC119 vector. The HpaI fragment between nucleotides 210 and
740 was replaced with the HIS3 gene, which was digested with
BamHI from pYAC3 vector and the end blunted with T4
polymerase. The resulting plasmid was cut with AccIII and
NcoI, and then the digest was transformed into MHY501. The
deletion of chromosomal ROS3 gene was confirmed by PCR
analysis using oligonucleotides specific to the 3'- and 5'-ends of the
open reading frame within ROS3 gene. The DNA amplification
product from the ROS3-deleted strain (ros3 Vesicle Preparation--
1-Myristoyl-2-(6-NBD-aminocaproyl)
phosphatidylethanolamine (C6-NBD-PE),
1-myristoyl-2-(6-NBD-aminocaproyl) phosphatidylcholine (C6-NBD-PC), and dioleoylphosphatidylcholine (DOPC) were
from Avanti Polar Lipids Inc. (Alabaster, AL).
1-Myristoyl-2-(6-NBD-aminocaproyl)phosphatidylserine (C6-NBD-PS) was synthesized from C6-NBD-PC by
phospholipase D (Seikagaku Corp., Tokyo, Japan)-catalyzed
transphosphatidylation (28) and purified. To prepare vesicles, lipids
were mixed in desired proportions (40 mol % C6-NBD-PE,
PC, or PS, 60 mol % DOPC), and chloroform was removed by evaporation
followed by vacuum desiccation. The resulting lipid film was
solubilized in SC medium, and the mixture was passed seven times
through a LiposoFast-Basic Stabilizer (Avestin, Inc., Ottawa,
Canada) equipped with 0.1-µm filters to produce evenly sized
vesicles. Total lipid concentration in the stock solution was 1 mM.
Internalization of Fluorescence-labeled Phospholipids into Yeast
Cells--
Fluorescence-labeled phospholipids internalization
experiments were performed as described by Kean et al. (9).
In brief, cultures in SD (A600 = 0.3-0.8) were
diluted to 107 cells/ml in fresh SD medium. Cells were then
incubated with vesicles containing 40%
C6-NBD-phospholipids and 60% DOPC (50 µM
total lipid concentration) with shaking for 30 min at 30 °C. For ATP depletion experiments, cells were resuspended in SCNaN3
containing required amino acids and incubated for 30 min at 30 °C
before addition of vesicles. After washing the cells three times with ice-cold SCNaN3 on ice, they were observed in a Zeiss
Axioplan microscope equipped with 100× Planneofluar oil immersion
objective (Carl Zeiss Co., Ltd., Oberkochen, Germany). Micrographs were taken with Fuji NEOPAN 1600 film. Flow cytometric analyses of the
internalization of C6-NBD-phospholipid into cells was
performed with a FACScan cytometer and CellQuest software (BD
PharMingen) as described previously (29).
Vital Dyes Uptake Studies--
FM4-64 and Lucifer yellow
carbohydrazide (LY-CH) were obtained from Molecular Probes Inc.
(Eugene, OR). Cell staining with these dyes was examined following the
methods by Vida and Emr (30) and Dulic and Riezman (31). A Zeiss
confocal laser scanning microscope using a 543-nm laser line for FM4-64
fluorescence and a 458-nm laser line for LY-CH was used for the
observation. Images were analyzed with LSM510 version 2.02 (Carl Zeiss
Co., Ltd.).
Activities of ABC Transporters--
Total RNA was extracted
using RNeasy Mini Kit (Qiagen) according to the manufacturer's
instructions. First strand cDNA was synthesized using
SuperScriptTM Choice System for cDNA Synthesis
(Invitrogen, Groningen, Netherlands) with the supplied
oligo(dT)12-18 primer. 25 ng of cDNA was used for each
subsequent PCR. For drug resistance assay, cycloheximide, 4-nitroquinoline-N-oxide, miconazole, and ketoconazole were
from Sigma. 10 mg/ml stock solutions were made by dissolving the drugs in dimethyl sulfoxide or ethanol. In the case of liquid medium, drug
sensitivity was determined as described above.
Protein Extraction and Endoglycosidase H
Treatment--
Harvested cells were washed in phosphate-buffered
saline and resuspended in SDS buffer containing 1/20th volume of
2-mercaptoethanol, and glass beads were added. Disruption of cells was
carried out with vortex mixer 7 times for 30 s, and solutions were
kept on ice. Lysates were boiled for 5 min, centrifuged to clear the
cell debris, and analyzed by 10% SDS-PAGE and Western blotting
analysis. Protein concentration was determined by BCA protein assay
reagent (Pierce). For endoglycosidase H treatment, the total cell
lysates were added to and equal volume of 0.15 M
citrate/phosphate buffer (pH 5.0) and 0.1 unit/ml endoglycosidase
(Seikagaku Corp.). The reaction mixtures were incubated for 20 h
at 37 °C.
Construction of EGFP-tagged ROS3--
To create a fusion protein
of Ros3p with EGFP, EGFP was digested with BamHI and
NotI from pEGFP-N1 (CLONTECH
Laboratories, Inc., Palo Alto, CA). The 3'-uncoding region of the
ROS3 gene was amplified from pS3 plasmid by PCR using an
upper primer added NotI site
(5'-TAATCATTGCGGCCGCAAAAGGAGTGATGGTTTTCTTTAT-3') and a lower primer T3
(5'-AATTAACCCTCACTAAAGGG-3'). The
NotI/KpnI-digested 3'-uncoding region was ligated
with BamHI/NotI-digested EGFP fragment and
subcloned to pRS416 vector (EGFP-3'-pRS416). The 5' promoter region of
ROS3 and the coding region, lacking stop codon (5'-ROS3*), was amplified from pS3 plasmid using an upper primer T7
(5'-GTAATACGACTCACTATAGGGC-3') and a lower primer added
BamHI site
(5'-GCTGGATCCGCTTTCATATTCCATGACAAACTTGA-3'). The
SalI/BamHI-digested 5'-ROS3* fragment was
inserted into the SalI/BamHI site of the
EGFP-3'-pRS416 plasmid. The resulting plasmid (5'-ROS3*-EGFP-3' in
pRS416) contained EGFP-fused Ros3p at its carboxyl terminus under the
control of the ROS3 promoter, and it was transformed into
the ros3 Subcellular Fractionation--
The plasma membrane was isolated
as described (32). The isolation of detergent-insoluble
glycolipid-enriched complexes (DIGs) was performed according to the
method described by Bagnat et al. (33) with some
modifications. In brief, mid-log phase cultures (equal to ~1 × 109 cells) were disrupted with glass beads. The lysates
were mixed with an equal volume of TNE buffer (25 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA)
containing 2% Triton X-100 and incubated for 30 min on ice. After
removal of cell debris, the detergent-treated lysates were added with
50% Opti-Prep (Sigma), TNE, 1% Triton X-100, to adjust to 35%
Opti-Prep, and overlaid with 30% Opti-Prep, TNE, 1% Triton X-100, and
TNE, 1% Triton X-100. The samples were centrifuged at 100,000 × g for 7.5 h at 4 °C in a Beckman SW55Ti rotor. Nine
fractions of equal volume were collected from the top, precipitated by
adding trichloroacetic acid, and resuspended in SDS buffer. Western
blotting analysis was performed as described above. Rabbit anti-Pma1p
was a gift from R. Serrano (Universidad Politecnica de Valencia,
Valencia, Spain). Rabbit anti-Gas1p, -Wbp1, and -Emp47p were provided
from H. Riezman (University of Basel, Basel, Switzerland). Mouse
anti-Por1p was purchased from Molecular Probes.
Isolation of the ros3 Mutant in a Screen for Mutants Hypersensitive
to Ro09-0198--
Ro09-0198 is a tetracyclic peptide that binds
specifically to PE in biological membranes and subsequently induces
cytolysis (18, 23). To isolate mutants with an altered PE organization on the plasma membrane, we selected a series of S. cerevisiae mutants that were hypersensitive to the peptide-induced
cytolysis. We screened ~55,000 mutagenized colonies and identified 17 ros (Ro sensitive) mutants. One of
the mutants, designated ros3, was most sensitive to the
peptide-induced cytolysis, and the LD50 of the peptide for
the ros3 mutant was approximately one-tenth compared with
wild type cells (Fig. 1A). There was no significant difference between the mutant and wild type
cells in their cellular phospholipid compositions (Table I) or in the sensitivities to various
agents such as amphotericin B, a polyene macrolide antibiotic that
binds to membrane ergosterol and induces cellular leakage (34) (Fig.
1B), detergents (SDS and Triton X-100), and ethanol (data
not shown). These results suggest that the hypersensitivity of the
ros3 mutant to the peptide resulted from an altered
organization of PE on the plasma membrane and not from an increased
cellular PE content nor from cell wall leakage caused by impaired cell
wall integrity.
Cloning of the ROS3 Gene--
The gene affected in the
ros3 mutant was cloned by complementation of the peptide
hypersensitive phenotype after transformation with a genomic library
(35). The DNA fragment that complemented the ros3 mutant
contains four ORFs (Fig. 2A).
Subcloning experiments indicated that the short fragment derived from
KpnI digestion was able to complement, but the other long
fragment was not (Fig. 2A). These results demonstrate that
YNL323W (GenBankTM accession number, Z71599),
which is also named as LEM3 (36) and BRE3 (37),
is the gene that complements the ros3 mutant. In this study,
we refer to the gene as ROS3. ROS3 encodes a protein of 414 amino acids that contains two putative transmembrane domains (Fig.
2B). Data base search revealed two genes with high
similarity to ROS3, CDC50 (GenBankTM
accession number, X59720, 40% identity in deduced amino acid sequence)
and YNR048W (GenBankTM accession number, Z71663, 38%
identity) in yeast genome (Fig. 2C). In addition, the
ROS3 genes are conserved in various organisms including worm
(GenBankTM accession number, U61953, 30% identity in
deduced amino acid sequence), fly (GenBankTM accession
number, AE003502, 33% identity), and mammals (GenBankTM
accession number, AA238841, 27% identity), suggesting that ROS3 homologs constitute a large gene family that has been
well conserved during evolution.
To examine further whether the ros3 mutant shows impaired
expression of the ROS3 gene product, we produced a
polyclonal antibody against a synthetic peptide corresponding to the
amino-terminal amino acids 1-19 of Ros3p. In immunoblotting, the
affinity-purified anti-Ros3p antibody bound specifically to a 62-kDa
protein in the wild type cells, whereas no protein band was detected in
the ros3 mutant (Fig.
3A). The observed relative
molecular mass was larger than that predicted by the primary structure
(47.4-kDa), but the treatment of cell lysate with endoglycosidase H
resulted in the reduction of molecular mass to 47.5-kDa which is fully compatible with the predicted mass (Fig. 3B). Because the
62-kDa protein was not present in the lysate of the
ROS3-deleted strain (see below) (Fig. 3A), these
results clearly demonstrate that the 62-kDa protein is Ros3p and that
the mutation of ros3 is caused by the defective expression
of Ros3p. Ros3p was not extracted with 1 M NaCl, 2 M urea, and 0.1 M NaCO3 (pH 11.0)
but was partially extracted with 1% (w/v) Triton X-100 (Fig.
3C). These results indicate that Ros3p is a glycosylated
transmembrane protein, which is consistent with the predicted structure
of the protein that has two putative membrane-spanning domains and six
possible glycosylation sites (Fig. 2B).
Ros3p Is Essential for Internalization of C6-NBD-PE and
C6-NBD-PC via a Non-Endocytic Pathway--
To examine the
cellular function of Ros3p, we disrupted the ROS3 gene in
S. cerevisiae by the replacement of a
531-bp internal HpaI fragment with 1,768-kb fragment
encoding the yeast HIS3 gene as shown in Fig.
4A. The deletion of
chromosomal ROS3 gene was confirmed by PCR analysis using
oligonucleotides specific to the 3'- and 5'-ends of the open reading
frame within ROS3. The ROS3-deleted strain
(ros3
To understand the role of Ros3p in PE organization in the plasma
membrane, we examined the transport and localization of
C6-NBD-PE, a fluorescence-labeled analog of PE molecule, in
the ros3 Ros3p Is Unrelated to Multidrug Resistance
Activity--
Overexpression of the yeast ABC transporters gives the
yeast cells a multidrug resistance phenotype, which results from the accelerated efflux of various amphipathic drugs (38, 39). It was shown
recently (9, 13) that some of the yeast ABC transporters, such as Pdr5p
and Yor1p, exhibit outward-directed phospholipid translocase activity.
To examine whether the reduced uptake of the fluorescence-labeled
phospholipids in the ros3 Subcellular Localization of Ros3p--
To assess the subcellular
localization of Ros3p, Ros3p tagged at the COOH terminus with EGFP was
introduced into the ros3
Bagnat et al. (33) recently isolated the detergent-insoluble
glycolipid-enriched complexes (DIGs) from yeast cells and showed that
DIGs were composed of phosphoinositol-based sphingolipids, ergosterol,
and glycosylphosphatidylinositol-anchored proteins, suggesting that
this yeast DIGs is identical to the lipid rafts of mammalian cells.
Because Ros3p was partially resistant to Triton X-100
extraction (Fig. 3C), we examined whether Ros3p was
localized in the yeast lipid rafts using the Opti-Prep density gradient (33, 44). As shown in Fig. 7C, a significant portion of
Ros3p was present in the low density DIGs fraction (fraction number 2 and 3) that was enriched with Gas1p and Pma1p, the major components of
the yeast lipid rafts (33, 44), whereas the non-DIG-associated protein
Wbp1p was absent from the DIGs fraction. These results suggest that a
part of Ros3p is associated with the yeast lipid rafts and raise the
possibility that Ros3p is involved in phospholipid translocation across
the bilayer membranes in the plasma membrane and the ER.
In the present study, we have screened yeast mutants that showed
hypersensitivity to the PE-binding antibiotic peptide, Ro09-0198. We
have isolated 17 clones of mutants and present our initial investigation of a mutant, named ros3, which showed the most
significant increase in the sensitivity to the peptide. The
ros3 mutant did not show any significant change in cellular
phospholipid composition or sensitivity to various agents such as
amphotericin B, a sterol-binding polyene macrolide, and detergents.
These data suggest that the ros3 mutation affects the PE
organization in the plasma membrane, rather than PE synthesis or
overall organization of the membrane structures. We first assumed that
the hypersensitivity of the ros3 mutant to the peptide
resulted from cell surface exposure of PE, which is normally localized
in the inner leaflet of the plasma membrane. Hence, we determined the
bilayer distribution of PE by using the membrane-impermeable reagent,
2,4,6-trinitrobenzenesulfonic acid, that chemically modifies the polar
head group of PE (45). We could, however, not detect more than a slight
increase in the 2,4,6-trinitrobenzenesulfonic acid-reactive PE in the
ros3 mutant compared with that observed with wild type cells
(data not shown). This could not account for the 10 times increase in
sensitivity to the peptide. The results suggested that the asymmetric
distribution of PE in the plasma membrane was not totally disrupted in
the ros3 mutant. Instead a disturbance in either the dynamic
movement or surface distribution of PE in the plasma membrane of the
ros3 mutated cells could cause the Ro09-0198
hypersensitivity. These observations have prompted us to undertake a
detailed analysis of the ros3 mutant. We identified the gene
defective in the ros3 mutant and studied the role of Ros3p
in the regulation of dynamic movement of PE in the plasma membrane.
Role of Ros3p in Transbilayer Movements of Phospholipids--
The
gene ROS3 was determined as a defective gene in the
ros3 mutant by the functional complementation screening of
the genomic library. The ROS3 gene product, Ros3p, was not
expressed in the ros3 mutant, and disruption of the
ROS3 gene caused the same hypersensitive phenotype as
observed in the ros3 mutant. These data further support that
the mutation of ros3 was caused by a defective expression of
the ROS3 gene product, Ros3p. We then demonstrated that
Ros3p is an essential component in the phospholipid translocation at the plasma membrane. The following lines of evidence support our conclusion. First, disruption of the ROS3 structural gene
resulted in a marked decrease in the uptake of the fluorescence-labeled PE analog, C6-NBD-PE (Fig. 5). Second, the uptake of both
soluble and lipophilic endocytic markers, Lucifer yellow and FM4-64,
was not affected in the ros3
Disruption of the ROS3 gene also caused total inhibition of
the uptake of C6-NBD-PC but showed no significant effect on
the uptake of C6-NBD-PS, suggesting that Ros3p is involved
in the transmembrane movement of PE and PC but not PS. Based
on the following discussions, we conclude that Ros3p is a novel type of
phospholipid transporter or a regulator of phospholipid translocation,
which is distinct from ABC transporters and P-type ATPase.
Two protein families have been implicated in phospholipid transport
across the yeast plasma membrane so far. The S. cerevisiae genome encodes 15 full-size putative ABC transporters, of which Pdr5p,
Yor1p, and Ste6p are suggested to exhibit outward-directed phospholipid
translocase activity as well as multidrug resistance activity (12, 13).
On the other hand, a novel P-type ATPase Drs2p is suggested to mediate
the inward-directed translocation of C6-NBD-PS in yeast
(14). Ros3p exhibits no significant homology with ABC transporters and
P-type ATPase (Fig. 2). Neither the expression levels nor the cellular
activities of ABC transporters were changed in the ros3
The transbilayer movements of the aminophospholipids such as PE and PS
across the plasma membrane is well established and appears to be a
common feature in eukaryotic cells. In contrast, an inward-directed
translocation of PC at the plasma membrane has only been observed in
few cell types, such as SV40-transformed WI-38 (46) and MDCK-II (47).
Transbilayer transport of C6-NBD-PC across the yeast plasma
membrane was also demonstrated previously (8-11). The translocation of
C6-NBD-PC, as well as C6-NBD-PE and C6-NBD-PS, in the yeast plasma membrane requires ATP and is
sensitive to sulfhydryl-modifying reagents. Ros3p is likely to mediate
translocation of both C6-NBD-PE and C6-NBD-PC,
but not C6-NBD-PS (Fig. 5), strongly suggesting that in
budding yeast C6-NBD-PE and C6-NBD-PC are
internalized by the same mechanism in which Ros3p is involved and that
C6-NBD-PS internalization is mediated by another
transporter, such as Drs2p.
Intracellular Localization of Ros3p and Its
Homolog--
Intracellular localization of EGFP-tagged Ros3p suggests
that Ros3p is localized in the ER membrane as well as in the plasma membrane (Fig. 7). There are several reports of protein-mediated phospholipid transport across yeast microsomal membrane (48) and
mammalian ER membrane (49-52). These translocase activities are less
specific for head group structure and do not require energy. Although
the molecular nature of ER phospholipid translocase has not been well
defined, it is possible that Ros3p may contribute to the transbilayer
movements of phospholipids in the ER membrane. Subcellular
fractionation studies indicate that a part of Ros3p is associated with
the yeast lipid rafts (Fig. 7). Because the yeast lipid rafts are
involved in protein sorting from ER to plasma membrane (33, 43), Ros3p
may be transported from ER to cell surface through the lipid rafts on
demand, though most of Ros3p is retained in the ER membrane during its
biosynthesis. Alternatively, Ros3p may be involved in phospholipid
translocation in the yeast lipid rafts.
Recent genetic analyses of yeast mutants suggest that the
ROS3 gene is involved in the glucocorticoid signal
transduction pathway (36) and in the brefeldin A sensitivity (37).
Although no obvious explanation for the correlations between these
functions of Ros3p has been obtained, these studies imply that the
mutation of the ROS3 gene affects diverse cellular
functions. The ROS3 gene shows a significant homology with
two other yeast genes, CDC50 and YNR048W (Fig. 2). Although
it has been reported (53) that the CDC50 mutation gives a
cold-sensitive phenotype, the functions of these Ros3p homologs are
unknown. Despite such remarkable similarities (~60% similarity in
amino acid level), deletion of either CDC50 or YNR048W had
little effect on the internalization of the C6-NBD
phospholipids from the plasma membrane (data not shown). It was shown
recently that Cdc50p appears to be predominantly localized in late
endosome membranes rather than in the ER or the plasma membrane, and
the cdc50 mutant shows defects in the uptake of
C6-NBD-PS, -PC, and -PE at low
temperature.2 Thus, it is
possible that the members of Ros3p protein family have distinct
characteristics of cellular localization and ability to regulate
phospholipid transport in various intracellular organelle membranes.
In conclusion, we have identified a novel membrane protein Ros3p
involved in the phospholipid translocation on the yeast plasma membrane. In addition, we have shown that Ros3p is localized in multiple organelles including the plasma membrane and the ER. Further
analyses of Ros3p and the ros3 We thank Dr. H. Riezman and Dr. R. Serrano
for the gift of antibodies; Drs. I. Yahara and S. Matsumoto for
providing the yeast expression vectors; and Drs. A. Nakano, K. Sato,
and K. Umebayashi for helpful suggestions.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Molecular
Biodynamics, the Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.:
81-3-3823-2105, ext.5430; Fax: 81-3-3823-2130; E-mail:
umeda@rinshoken.or.jp.
Published, JBC Papers in Press, July 19, 2002, DOI 10.1074/jbc.M205564200
2
K. Fujimura-Kamada and K. Tanaka, personal communication.
The abbreviations used are:
PE, phosphatidylethanolamine;
Ro, Ro09-0198;
PC, phosphatidylcholine;
PS, phosphatidylserine;
NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl;
C6-NBD-PE, 1-myristoyl-2(6-NBD-aminocaproyl)-PE;
C6-NBD-PC, 1-myristoyl-2(6-NBD-aminocaproyl)-PC;
C6-NBD-PS, 1-myristoyl-2(6-NBD-aminocaproyl)-PS;
DOPC, dioleoylphosphatidylcholine;
FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium
dibromide;
ABC transporter, ATP-binding cassette transporter;
EGFP, the
red-shifted green fluorescence protein variant;
LY-CH, Lucifer yellow
carbohydrazide;
DIGs, detergent-insoluble glycolipid-enriched
complexes;
ORF, open reading frame;
ER, endoplasmic reticulum.
A Novel Membrane Protein, Ros3p, Is Required for Phospholipid
Translocation across the Plasma Membrane in Saccharomyces
cerevisiae*
§,,,
,**
Department of Molecular Biodynamics, the
Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, the § Department of Biology,
Faculty of Science, Ochanomizu University, 2-1-1 Ohtsuka,
Bunkyo-ku, Tokyo 112-8610, Supra-Biomolecular System Research
Group, RIKEN (Institute of Physical and Chemical Research), Frontier
Research System, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, and the
¶ Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 detergent-insoluble 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells in the translocation of
fluorescence-labeled phospholipids across the plasma membrane (10, 15).
Thus, at present, the molecular identity of the proteins involved in
the inward-directed translocation of phospholipids still remains unknown.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
his3 leu2 lys2 trp1
ura3) was used for the construction of strains deleted for the
ROS3 gene. The media used are as follows: YPD, medium
containing 1% yeast extract, 2% polypeptone, and 2% glucose; SD,
medium containing 0.17% yeast nitrogen base without amino acids and
ammonium sulfate, 0.5% ammonium sulfate, 2% glucose, and the required
amino acids; SG, SD lacking glucose but containing 2% galactose; SC,
SD lacking glucose but containing 2% sorbitol; SCNaN3, SC
containing 20 mM sodium azide. Unless otherwise noted, the
indicated strains were grown to early to mid-log phase at 30 °C.
Yeast transformations were performed by the lithium acetate method
(24).
strain) was ~1.2-kb pair larger than the product obtained from the
parental strain. The PCR product from the ros3 strain was digested by HindIII into three fragments, whereas the
product from the wild type was not digested (Fig. 4A),
confirming that the ROS3::HIS3 fragment
was integrated into the predicted site of chromosome.
strain. For microscopy, Ros3p-EGFP strain was
cultured to early log phase at 20 or 30 °C and observed with Zeiss
confocal laser scanning microscope using a 488-nm laser line. Images
were analyzed with LSM510 version 2.02 (Carl Zeiss).
RESULTS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sensitivity of the ros3
mutant to the Ro peptide and amphotericin B. Mid-log phase
cells were diluted to 1 × 106 cells/ml in YPD
containing Ro peptide (A) or amphotericin B (B)
at the indicated concentrations. After a 24-h incubation,
A600 values were measured, and the value of the
0 µM culture was normalized to 100% viability. There was
no difference in growth rate between the wild type ( 
) and the
ros3 mutant (
) cultured without the additives. Each point
represents the means and S.D. of three independent experiments.
Phospholipid composition of ros3 mutant strainsa
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Fig. 2.
The complementary gene of the ros3
mutant. A, pS3 plasmid, the genomic clone that
complemented the ros3 mutation. The black arrows
depict the position and direction of the ORFs within this region. The
solid line indicates yeast genomic region in the pS3
plasmid, and dashed line shows YCp50 plasmid region.
S, SalI site; K, KpnI site;
H, HindIII site. B, amino acid
sequence of Ros3p. The putative transmembrane domains are underlined.
The dotted lines indicate the putative
N-glycosylation sites. The amino-terminal peptide
(boxed) was synthesized and used in the generation of
anti-Ros3p antibody. C, alignment of amino acid sequences of
Ros3p, Cdc50p, and the hypothetical ORF product, Ynr048p. Identical
amino acids in two or more sequences are highlighted on a black
background.
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Fig. 3.
Defective expression of Ros3p in the
ros3 mutant and biochemical characterization of
Ros3p. A, total cellular proteins from wild type
(WT) cells, the ros3 mutant, and
ROS3-deleted cells (ros3 strain, see
"Experimental Procedures") were subjected to SDS-PAGE and analyzed
by immunoblotting with affinity-purified rabbit anti-Ros3p polyclonal
antibody. B, the cellular proteins were treated with
endoglycosidase H (Endo H) at 37 °C for 20 h and
analyzed by immunoblotting using the anti-Ros3p antibody. C,
the total cellular homogenates were treated with various chemical
reagents as indicated at top and centrifuged at 100,000 × g for 30 min. Soluble (S) and particulate
(P) fractions were derived from the same amount of the
homogenates. TX-100, Triton X-100. Ros3p was detected by
immunoblotting.
strain) could not grow on the YPD plate containing 10 µM Ro peptide, and the ROS3 gene on a
single copy plasmid suppressed this sensitivity (Fig. 4B).
The ros3 strain also showed similar phospholipid
composition (Table I) and amphotericin B sensitivity with those
observed with the ros3 mutant (data not shown).
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Fig. 4.
Sensitivity of the ros3 strain
to the Ro peptide. A, the disruption of ROS3 with
HIS3 gene. The protein-coding regions are shown by
open arrows. A, AccIII site;
Hp, HpaI site; N, NcoI
site; B, BamHI site; H,
HindIII site. B, single colonies of the wild type
(WT), the ros3 mutant, the ros3
strain (ros3), and the ros3 strain
transformed with a single copy of the ROS3 gene
(ros3 + ROS3) were transferred to YPD plates
with or without 10 µM Ro peptide and cultured at 30 for
~2 days.
strain. It was shown previously that yeast cells
incubated with lipid vesicles containing C6-NBD-PE
internalized the fluorescence-labeled lipids from the outer leaflet of
the plasma membrane (8-10, 15). When the cells were incubated at
30 °C in the presence of vesicles containing 40 mol %
C6-NBD-PE and 60 mol % DOPC, the intracellular organelles
of the wild type cells were strongly labeled, whereas the intracellular
fluorescence of the ros3 strain was much fainter (Fig.
5A, panels a and
b). Phospholipid extraction and separation by thin layer
chromatography revealed no degradation products of
C6-NBD-PE in the cell extract (data not shown), indicating that only intact C6-NBD-PE was internalized. Flow
cytometric analyses showed that the uptake of C6-NBD-PE in
the ros3 strain was reduced to ~10% that of the wild
type cells (Fig. 5B). In the ros3 strain, the
uptake of C6-NBD-PC was also significantly decreased,
whereas no significant change in the uptake of C6-NBD-PS
was observed (Fig. 5B). Depletion of intracellular ATP
reduced the uptake of C6-NBD-PE in the wild type cells to
the level of the ros3 strain (Fig. 5B),
suggesting that the Ros3p is responsible for the
ATP-dependent uptake of the fluorescence-labeled
phospholipids. The uptake and intracellular distribution of the
endocytic markers, Lucifer yellow (31) and FM4-64 (30), was not
affected in the ros3 strain (Fig. 5A,
panels d and f), indicating that the
ros3 strain was not defective in endocytosis of soluble
and amphipathic molecules. Because the expression of the
ROS3 gene by using centromeric plasmid restored the uptake
of both C6-NBD-PE and C6-NBD-PC of the
ros3 strain (Fig. 5B), these results suggest
that Ros3p is required for the translocation of both
C6-NBD-PE and C6-NBD -PC across the plasma
membrane through the non-endocytic pathway.
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Fig. 5.
Internalization of fluorescence-labeled
analogs of phospholipids. A, internalization of
C6-NBD-PE and endocytic markers was examined as described
under "Experimental Procedures." The cells were incubated with
C6-NBD-PE (panels a and b), FM4-64
(panels c and d), or Lucifer yellow
(LY-CH) (panels e and f) at 30 °C.
Left panels a, c, and e,
wild type (WT) cell; right panels b,
d, and f, ros3 strain. B,
cells were incubated with C6-NBD-PE, -PC, or -PS, and the
intensity of fluorescence-labeled phospholipids within the cells was
analyzed with a FACScan cytometer. Top, wild type cell;
middle, ros3 strain; bottom,
ros3 strain transformed with a single copy plasmid
containing ROS3 gene.
strain resulted from the
enhanced outward-directed translocation of the phospholipid analogs
through ABC transporters, we investigated the mRNA levels of yeast
ABC transporters as well as the sensitivity of the ros3
strain against various cytotoxic compounds that are shown to be
substrates of the yeast ABC transporters (40, 41). Reverse
transcriptase-PCR analyses showed no significant difference in the
mRNA levels of both Pdr5p and Yor1p between the ros3
strain and wild type cells (Fig.
6A). In addition, there was no
significant difference between the ros3 strain and wild
type cells in the sensitivities against various amphipathic cytotoxic
drugs, such as miconazole, ketoconazole,
4-nitroquinoline-N-oxide, and cycloheximide (Fig. 6,
B-E). These results suggest that the uptake deficiency of
the fluorescence-labeled phospholipids in the ros3 strain is not caused by the enhanced outward movement of the lipids mediated by the multidrug resistance activity of the transporters.
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Fig. 6.
Expression of ABC transporters and multidrug
resistance activities in the ros3 strain.
A, cells were grown in SD medium. mRNA levels of
ROS3, PDR5, and YOR1 in wild type
cells (WT, left) and the ros3
strain (right) were examined by reverse transcriptase-PCR
analyses as described under "Experimental Procedures."
B-E, cells grown in YPD medium were diluted to 1 × 106 cells/ml in YPD medium containing cycloheximide
(B), 4-nitroquinoline-N-oxide (C),
miconazole (D), or ketoconazole (E) at indicated
concentrations and incubated for 24 h. Viabilities of the cells
were determined by measuring A600 values.
strain. Chimeric protein
Ros3p-EGFP complemented the Ro peptide sensitivity phenotype of the
ros3 strain on the centromeric plasmid (data not shown),
indicating that Ros3p-EGFP is fully functional. Logarithmically growing
cells expressing Ros3p-EGFP by its own promoter showed the fluorescent
staining depicted in Fig.
7A, panel a.
Ros3p-EGFP was uniformly localized in the nuclear periphery and in
discrete patches associated with the cytoplasmic membrane. This pattern
coincides with the typical ER markers such as Sec63p (42). In addition
to ER, a part of Ros3p-EGFP appeared to be localized at the plasma
membrane (Fig. 7A, panel b). To examine further
Ros3p localization, a fraction enriched in plasma membranes was
prepared by sucrose density gradient fractionation. Ros3p was
concentrated in the plasma membrane fraction, which was enriched for
the plasma membrane marker protein Pma1p. Other organelle marker
proteins including Wbp1p (ER), Emp47p (Golgi), and Por1p (mitochondria)
distributed differently in the sucrose gradient (Fig. 7B).
The results indicate that Ros3p is localized in both the plasma
membrane and the ER membrane.
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Fig. 7.
Subcellular localization of Ros3p.
A, the ros3 strain expressing Ros3p-EGFP was
grown to early log phase at 20 °C, and observed by confocal
fluorescence microscopy. Arrow (panel a) and
arrowhead (panel b) indicate the ER and the
plasma membrane staining, respectively. B, the plasma
membrane fraction was purified as described under "Experimental
Procedures." The total cellular lysate (L) and plasma
membrane fraction (PM) were subjected to SDS-PAGE, followed
by immunoblotting analyses with anti-Ros3p, anti-Pma1p (plasma membrane
marker), anti-Wbp1 (ER marker), anti-Emp47p (Golgi marker), and
anti-Por1p (mitochondria marker). C, detergent-insoluble
glycolipid-enriched complexes (DIGs) were isolated as
described under "Experimental Procedures." The presence of Ros3p
and marker proteins Gas1p and Pma1p, for the major DIG-associated
proteins, and Wbp1p for non-DIG-associated protein in each fraction
collected from the top of the Opti-Prep density gradient was examined
by immunoblotting.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
strain (Fig. 5). This
indicates that Ros3p is not involved in the lipid internalization via
the conventional endocytic pathway. Finally, a significant portion of
Ros3p is localized in the plasma membrane (Fig. 7). It has been shown
that ATP depletion treatment at low temperature impairs the
C6-NBD-phospholipid internalization from the outer leaflet
of the yeast plasma membrane but not insertion into the outer leaflet
after transport from the donor vesicles (9, 11). In the present study,
we also confirmed that the C6-NBD-PE internalization was
ATP-dependent. As the disruption of the ROS3
gene greatly reduced the uptake of C6-NBD-PE, it is likely
that Ros3p is responsible for the ATP-dependent internalization of the lipid analogs from the outer leaflet of plasma
membrane, rather than the ATP-independent insertion of lipid analogs
into the outer leaflet of plasma membrane. Taken together, Ros3p is
likely to play a role in the phospholipid translocation across the
plasma membrane, probably by the transbilayer transport.
strain (Fig. 6), suggesting that Ros3p is unrelated to ABC
transporters. Furthermore, C6-NBD-PS internalization was
not affected by the loss of Ros3p function (Fig. 5), and Ros3p function
is thereby not related to Drs2p.
strain will help us
understand the regulation of the phospholipid translocation across the
bilayer membranes as well as its role in the regulation of various
cellular functions.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1.
Devaux, P. F.
(1991)
Biochemistry
30,
1163-1173[CrossRef][Medline]
[Order article via Infotrieve]
2.
Schroit, A. J.,
and Zwaal, R. F.
(1991)
Biochim. Biophys. Acta
1071,
313-329[Medline]
[Order article via Infotrieve]
3.
Zachowski, A.
(1993)
Biochem. J.
294,
1-14
4.
Bevers, E. M.,
Comfurius, P.,
Dekkers, D. W.,
and Zwaal, R. F.
(1999)
Biochim. Biophys. Acta
1439,
317-330[Medline]
[Order article via Infotrieve]
5.
Martin, O. C.,
and Pagano, R. E.
(1987)
J. Biol. Chem.
262,
5890-5898 6.
Smith, A. J.,
Timmermans-Hereijgers, J. L.,
Roelofsen, B.,
Wirtz, K. W.,
van Blitterswijk, W. J.,
Smit, J. J.,
Schinkel, A. H.,
and Borst, P.
(1994)
FEBS Lett.
354,
263-266[CrossRef][Medline]
[Order article via Infotrieve]
7.
van Helvoort, A.,
Smith, A. J.,
Sprong, H.,
Fritzsche, I.,
Schinkel, A. H.,
Borst, P.,
and van Meer, G.
(1996)
Cell
87,
507-517[CrossRef][Medline]
[Order article via Infotrieve]
8.
Kean, L. S.,
Fuller, R. S.,
and Nichols, J. W.
(1993)
J. Cell Biol.
123,
1403-1419 9.
Kean, L. S.,
Grant, A. M.,
Angeletti, C.,
Mahe, Y.,
Kuchler, K.,
Fuller, R. S.,
and Nichols, J. W.
(1997)
J. Cell Biol.
138,
255-270 10.
Marx, U.,
Polakowski, T.,
Pomorski, T.,
Lang, C.,
Nelson, H.,
Nelson, N.,
and Herrmann, A.
(1999)
Eur. J. Biochem.
263,
254-263[Medline]
[Order article via Infotrieve]
11.
Grant, A. M.,
Hanson, P. K.,
Malone, L.,
and Nichols, J. W.
(2001)
Traffic
2,
37-50[CrossRef][Medline]
[Order article via Infotrieve]
12.
Ruetz, S.,
Brault, M.,
Dalton, W.,
and Gros, P.
(1997)
Biochemistry
36,
8180-8188[CrossRef][Medline]
[Order article via Infotrieve]
13.
Decottignies, A.,
Grant, A. M.,
Nichols, J. W.,
de Wet, H.,
McIntosh, D. B.,
and Goffeau, A.
(1998)
J. Biol. Chem.
273,
12612-12622 14.
Tang, X.,
Halleck, M. S.,
Schlegel, R. A.,
and Williamson, P.
(1996)
Science
272,
1495-1497[Abstract]
15.
Siegmund, A.,
Grant, A.,
Angeletti, C.,
Malone, L.,
Nichols, J. W.,
and Rudolph, H. K.
(1998)
J. Biol. Chem.
273,
34399-34405 16.
Wakamatsu, K.,
Choung, S. Y.,
Kobayashi, T.,
Inoue, K.,
Higashijima, T.,
and Miyazawa, T.
(1990)
Biochemistry
29,
113-118[CrossRef][Medline]
[Order article via Infotrieve]
17.
Umeda, M.,
and Emoto, K.
(1999)
Chem. Phys. Lipids
101,
81-91[CrossRef][Medline]
[Order article via Infotrieve]
18.
Aoki, Y.,
Uenaka, T.,
Aoki, J.,
Umeda, M.,
and Inoue, K.
(1994)
J. Biochem. (Tokyo)
116,
291-297 19.
Emoto, K.,
Toyama-Sorimachi, N.,
Karasuyama, H.,
Inoue, K.,
and Umeda, M.
(1997)
Exp. Cell Res.
232,
430-434[CrossRef][Medline]
[Order article via Infotrieve]
20.
Emoto, K.,
Kobayashi, T.,
Yamaji, A.,
Aizawa, H.,
Yahara, I.,
Inoue, K.,
and Umeda, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12867-12872 21.
Emoto, K.,
and Umeda, M.
(2000)
J. Cell Biol.
149,
1215-1224 22.
Bogdanov, M.,
Umeda, M.,
and Dowhan, W.
(1999)
J. Biol. Chem.
274,
12339-12345 23.
Emoto, K.,
Kuge, O.,
Nishijima, M.,
and Umeda, M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12400-12405 24.
Ito, H.,
Fukuda, Y.,
Murata, K.,
and Kimura, A.
(1983)
J. Bacteriol.
153,
163-168 25.
Lawrence, C. W.
(1991)
Methods Enzymol.
194,
273-281[Medline]
[Order article via Infotrieve]
26.
Satoh, O.,
Umeda, M.,
Imai, H.,
Tunoo, H.,
and Inoue, K.
(1990)
J. Lipid Res.
31,
1293-1300[Abstract]
27.
Harada, A.,
Furuta, B.,
Takeuchi, K.,
Itakura, M.,
Takahashi, M.,
and Umeda, M.
(2000)
J. Biol. Chem.
275,
36885-36891 28.
Kato, S.,
Kokusho, Y.,
and Machida, H.
(1984)
Agric. Biol. Chem.
48,
2181-2188
29.
Hanson, P. K.,
and Nichols, J. W.
(2001)
J. Biol. Chem.
276,
9861-9867 30.
Vida, T. A.,
and Emr, S. D.
(1995)
J. Cell Biol.
128,
779-792 31.
Dulic, V.,
and Riezman, H.
(1990)
J. Cell Sci.
97,
517-525 32.
Serrano, R.
(1988)
Methods Enzymol.
157,
533-544[Medline]
[Order article via Infotrieve]
33.
Bagnat, M.,
Keranen, S.,
Shevchenko, A.,
and Simons, K.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3254-3259 34.
Brajtburg, J.,
Powderly, W. G.,
Kobayashi, G. S.,
and Medoff, G.
(1990)
Antimicrob. Agents Chemother.
34,
183-188 35.
Rose, M. D.
(1987)
Methods Enzymol.
152,
481-504[Medline]
[Order article via Infotrieve]
36.
Sitcheran, R.,
Emter, R.,
Kralli, A.,
and Yamamoto, K. R.
(2000)
Genetics
156,
963-972 37.
Muren, E.,
Oyen, M.,
Barmark, G.,
and Ronne, H.
(2001)
Yeast
18,
163-172[CrossRef][Medline]
[Order article via Infotrieve]
38.
Higgins, C. F.
(1992)
Annu. Rev. Cell Biol.
8,
67-113[CrossRef]
39.
Gottesman, M. M.,
and Pastan, I.
(1993)
Annu. Rev. Biochem.
62,
385-427[CrossRef][Medline]
[Order article via Infotrieve]
40.
Balzi, E.,
and Goffeau, A.
(1994)
Biochim. Biophys. Acta
1187,
152-162[Medline]
[Order article via Infotrieve]
41.
van den Hazel, H. B.,
Pichler, H.,
do Valle Matta, M. A.,
Leitner, E.,
Goffeau, A.,
and Daum, G.
(1999)
J. Biol. Chem.
274,
1934-1941 42.
Sadler, I.,
Chiang, A.,
Kurihara, T.,
Rothblatt, J.,
Way, J.,
and Silver, P.
(1989)
J. Cell Biol.
109,
2665-2675 43.
Muniz, M.,
Morsomme, P.,
and Riezman, H.
(2001)
Cell
104,
313-320[CrossRef][Medline]
[Order article via Infotrieve]
44.
Bagnat, M.,
Chang, A.,
and Simons, K.
(2001)
Mol. Biol. Cell
12,
4129-4138 45.
Kobayashi, T.,
and Pagano, R. E.
(1989)
J. Biol. Chem.
264,
5966-5973 46.
Sleight, R. G.,
and Abanto, M. N.
(1989)
J. Cell Sci.
93,
363-374 47.
Pomorski, T.,
Herrmann, A.,
Muller, P.,
van Meer, G.,
and Burger, K.
(1999)
Biochemistry
38,
142-150[CrossRef][Medline]
[Order article via Infotrieve]
48.
Nicolson, T.,
and Mayinger, P.
(2000)
FEBS Lett.
476,
277-281[CrossRef][Medline]
[Order article via Infotrieve]
49.
Bishop, W. R.,
and Bell, R. M.
(1985)
Cell
42,
51-60[CrossRef][Medline]
[Order article via Infotrieve]
50.
Backer, J. M.,
and Dawidowicz, E. A.
(1987)
Nature
327,
341-343[CrossRef][Medline]
[Order article via Infotrieve]
51.
Herrmann, A.,
Zachowski, A.,
and Devaux, P. F.
(1990)
Biochemistry
29,
2023-2027[CrossRef][Medline]
[Order article via Infotrieve]
52.
Menon, A. K.,
Watkins, W. E. R.,
and Hrafnsdottir, S.
(2000)
Curr. Biol.
10,
241-252[CrossRef][Medline]
[Order article via Infotrieve]
53.
Moir, D.,
Stewart, S. E.,
Osmond, B. C.,
and Botstein, D.
(1982)
Genetics
100,
547-563
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H. C. Stevens, L. Malone, and J. W. Nichols The Putative Aminophospholipid Translocases, DNF1 and DNF2, Are Not Required for 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-phosphatidylserine Flip across the Plasma Membrane of Saccharomyces cerevisiae J. Biol. Chem., December 12, 2008; 283(50): 35060 - 35069. [Abstract] [Full Text] [PDF]
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 M. Ikeda, A. Kihara, A. Denpoh, and Y. Igarashi The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry Mol. Biol. Cell, May 1, 2008; 19(5): 1922 - 1931. [Abstract] [Full Text] [PDF]
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K. Nakano, T. Yamamoto, T. Kishimoto, T. Noji, and K. Tanaka Protein Kinases Fpk1p and Fpk2p are Novel Regulators of Phospholipid Asymmetry Mol. Biol. Cell, April 1, 2008; 19(4): 1783 - 1797. [Abstract] [Full Text] [PDF]
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 L. R. Poulsen, R. L. Lopez-Marques, S. C. McDowell, J. Okkeri, D. Licht, A. Schulz, T. Pomorski, J. F. Harper, and M. G. Palmgren The Arabidopsis P4-ATPase ALA3 Localizes to the Golgi and Requires a {beta}-Subunit to Function in Lipid Translocation and Secretory Vesicle Formation PLANT CELL, March 1, 2008; 20(3): 658 - 676. [Abstract] [Full Text] [PDF]
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D. L. Daleke Phospholipid Flippases J. Biol. Chem., January 12, 2007; 282(2): 821 - 825. [Full Text] [PDF]
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N. Furuta, K. Fujimura-Kamada, K. Saito, T. Yamamoto, and K. Tanaka Endocytic Recycling in Yeast Is Regulated by Putative Phospholipid Translocases and the Ypt31p/32p-Rcy1p Pathway Mol. Biol. Cell, January 1, 2007; 18(1): 295 - 312. [Abstract] [Full Text] [PDF]
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W. R. Riekhof and D. R. Voelker Uptake and Utilization of Lyso-phosphatidylethanolamine by Saccharomyces cerevisiae J. Biol. Chem., December 1, 2006; 281(48): 36588 - 36596. [Abstract] [Full Text] [PDF]
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F. J. Perez-Victoria, M. P. Sanchez-Canete, S. Castanys, and F. Gamarro Phospholipid Translocation and Miltefosine Potency Require Both L. donovani Miltefosine Transporter and the New Protein LdRos3 in Leishmania Parasites J. Biol. Chem., August 18, 2006; 281(33): 23766 - 23775. [Abstract] [Full Text] [PDF]
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 J. M. Cerutti, F. R.M. Latini, C. Nakabashi, R. Delcelo, V. P. Andrade, M. J. Amadei, R. M.B. Maciel, F. C. Hojaij, D. Hollis, J. Shoemaker, et al. Diagnosis of Suspicious Thyroid Nodules Using Four Protein Biomarkers. Clin. Cancer Res., June 1, 2006; 12(11): 3311 - 3318. [Abstract] [Full Text] [PDF]
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S. M. Elvington, F. Bu, and J. W. Nichols Fluorescent, Acyl Chain-labeled Phosphatidylcholine Analogs Reveal Novel Transport Pathways across the Plasma Membrane of Yeast J. Biol. Chem., December 9, 2005; 280(49): 40957 - 40964. [Abstract] [Full Text] [PDF]
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A. Kihara and Y. Igarashi Cross Talk between Sphingolipids and Glycerophospholipids in the Establishment of Plasma Membrane Asymmetry Mol. Biol. Cell, November 1, 2004; 15(11): 4949 - 4959. [Abstract] [Full Text] [PDF]
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 K. Iwamoto, S. Kobayashi, R. Fukuda, M. Umeda, T. Kobayashi, and A. Ohta Local exposure of phosphatidylethanolamine on the yeast plasma membrane is implicated in cell polarity Genes Cells, October 1, 2004; 9(10): 891 - 903. [Abstract] [Full Text] [PDF]
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 P. Natarajan, J. Wang, Z. Hua, and T. R. Graham Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function PNAS, July 20, 2004; 101(29): 10614 - 10619. [Abstract] [Full Text] [PDF]
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K. Saito, K. Fujimura-Kamada, N. Furuta, U. Kato, M. Umeda, and K. Tanaka Cdc50p, a Protein Required for Polarized Growth, Associates with the Drs2p P-Type ATPase Implicated in Phospholipid Translocation in Saccharomyces cerevisiae Mol. Biol. Cell, July 1, 2004; 15(7): 3418 - 3432. [Abstract] [Full Text] [PDF]
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 T. Pomorski, J. C. M. Holthuis, A. Herrmann, and G. van Meer Tracking down lipid flippases and their biological functions J. Cell Sci., February 22, 2004; 117(6): 805 - 813. [Abstract] [Full Text] [PDF]
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F. J. Perez-Victoria, F. Gamarro, M. Ouellette, and S. Castanys Functional Cloning of the Miltefosine Transporter: A NOVEL P-TYPE PHOSPHOLIPID TRANSLOCASE FROM LEISHMANIA INVOLVED IN DRUG RESISTANCE J. Biol. Chem., December 12, 2003; 278(50): 49965 - 49971. [Abstract] [Full Text] [PDF]
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P. K. Hanson, L. Malone, J. L. Birchmore, and J. W. Nichols Lem3p Is Essential for the Uptake and Potency of Alkylphosphocholine Drugs, Edelfosine and Miltefosine J. Biol. Chem., September 19, 2003; 278(38): 36041 - 36050. [Abstract] [Full Text] [PDF]
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 F. J. Perez-Victoria, S. Castanys, and F. Gamarro Leishmania donovani Resistance to Miltefosine Involves a Defective Inward Translocation of the Drug Antimicrob. Agents Chemother., August 1, 2003; 47(8): 2397 - 2403. [Abstract] [Full Text] [PDF]
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T. Pomorski, R. Lombardi, H. Riezman, P. F. Devaux, G. van Meer, and J. C. M. Holthuis Drs2p-related P-type ATPases Dnf1p and Dnf2p Are Required for Phospholipid Translocation across the Yeast Plasma Membrane and Serve a Role in Endocytosis Mol. Biol. Cell, March 1, 2003; 14(3): 1240 - 1254. [Abstract] [Full Text] [PDF]
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K. Misu, K. Fujimura-Kamada, T. Ueda, A. Nakano, H. Katoh, and K. Tanaka Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae Mol. Biol. Cell, February 1, 2003; 14(2): 730 - 747. [Abstract] [Full Text] [PDF]
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A. Makino, T. Baba, K. Fujimoto, K. Iwamoto, Y. Yano, N. Terada, S. Ohno, S. B. Sato, A. Ohta, M. Umeda, et al. Cinnamycin (Ro 09-0198) Promotes Cell Binding and Toxicity by Inducing Transbilayer Lipid Movement J. Biol. Chem., January 24, 2003; 278(5): 3204 - 3209. [Abstract] [Full Text] [PDF]
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