Molecular Biology of the Cell
Vol. 20, 2920–2931, June 15, 2009
Control of Protein and Sterol Trafficking by Antagonistic
Activities of a Type IV P-type ATPase and Oxysterol
Binding Protein Homologue
Baby-Periyanayaki Muthusamy,* Sumana Raychaudhuri,†
Paramasivam Natarajan,* Fumiyoshi Abe,‡Ke Liu,* William A. Prinz,†
and Todd R. Graham*
*Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634;†Laboratory of Cell
Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD 20892; and‡Extremobiosphere Research Center, Japan Agency for Marine-
Earth Science and Technology (JAMSTEC), Yokosuka 237-0061, Japan
Submitted October 16, 2008; Accepted April 16, 2009
Monitoring Editor: Akihiko Nakano
The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces
cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an
unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type
ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs)
growth defect of drs2?, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p.
Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes
Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN
membranes from kes1? cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also
antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the
plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular
membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which
appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid orga-
nization that resists sterol extraction by a sterol binding protein.
A fundamental feature of the eukaryotic cell plasma mem-
brane is the asymmetric distribution of phospholipid species
between the inner and outer leaflets. Phosphatidylserine
(PS) and phosphatidylethanolamine (PE) are highly concen-
trated in the inner, cytosolic leaflet, with sphingolipids and
phosphatidylcholine (PC) enriched in the extracellular leaf-
let (Balasubramanian and Schroit, 2003). The mechanism for
establishing and maintaining phospholipid asymmetry is
uncertain; however, type IV P-type ATPases (P4-ATPases)
seem to facilitate this process by flipping PS and PE to the
cytosolic leaflet (Graham, 2004; van Meer et al., 2008). The
budding yeast genome contains five P4-ATPase genes—
DRS2, DNF1, DNF2, DNF3, and NEO1—whereas the human
genome contains 14 members of this gene family (Graham,
2004). NEO1 is an essential gene and the other four members
(DRS2 and the DNF genes) form an essential group with
partially overlapping functions. Drs2p localizes to the trans-
Golgi network (TGN) and is required for a flippase activity
present in purified TGN membranes with specificity for PS
and PE (Natarajan et al., 2004; Alder-Baerens et al., 2006).
Dnf1p and Dnf2p seem to have a substrate preference for PE
and PC and are primarily responsible for flippase activity in
the yeast plasma membrane (Pomorski et al., 2003; Saito et al.,
2004; Elvington et al., 2005). Most of the P4-ATPases interact
with a noncatalytic subunit in the Cdc50p family. For exam-
ple, Drs2p associates with Cdc50p, whereas Dnf1p and
Dnf2p associate with Lem3p (also called Ros3p), and these
interactions are required for export of the complexes out of
the endoplasmic reticulum (ER) and the function of the
P4-ATPase (Saito et al., 2004; Chen et al., 2006).
Another well-conserved feature of the eukaryotic plasma
membrane is its high concentration of sterol relative to in-
ternal organelles. Even though different species produce
unique sterols, for example cholesterol in mammals and
ergosterol in fungi, the majority of unesterified cellular ste-
rol is localized to the plasma membrane (Daum et al., 1998;
Liscum and Munn, 1999). How sterols are concentrated in
the plasma membrane is poorly understood. Surprisingly,
sterols can move efficiently between the ER, the site of
synthesis, and plasma membrane under conditions in which
vesicle-mediated protein transport through the secretory
pathway is blocked (Kaplan and Simoni, 1985; Li and Prinz,
2004; Baumann et al., 2005). Candidate proteins responsible
for this nonvesicular, intracellular sterol transport include
the oxysterol binding protein homologues (Osh proteins).
These sterol binding proteins include seven members in
This article was published online ahead of print in MBC in Press
on April 29, 2009.
Address correspondence to: Todd R. Graham (tr.graham@
2920 © 2009 by The American Society for Cell Biology
Saccharomyces cerevisiae (Osh1p-Osh7p) and 16 human ho-
mologues (Beh et al., 2001). The structure of Osh4p/Kes1p
(hereafter referred to as Kes1p) has been solved with sterol
present in a deep, hydrophobic binding pocket that is sim-
ilar to that present in other lipid transfer proteins (Im et al.,
2005). Kes1p can transfer sterol between membranes in vitro
and inactivation of Kes1p in a strain deficient for the other
six Osh proteins perturbs nonvesicular sterol transport in
vivo (Im et al., 2005; Raychaudhuri et al., 2006). However,
whether Osh proteins mediate bulk sterol transport or func-
tion as signaling molecules in response to sterol binding is
an important unresolved issue (Fairn and McMaster, 2008).
P4-ATPases and oxysterol binding proteins have also been
implicated in vesicle-mediated protein transport (Graham,
2004; Mousley et al., 2007). DRS2 was recovered in a genetic
screen for factors that function with ADP-ribosylation factor
(ARF) in vesicle biogenesis from the Golgi (Chen et al., 1999).
This screen also recovered CDC50 (Chen et al., 2006), which
encodes a membrane protein that chaperones Drs2p from
the ER to the TGN (Saito et al., 2004), the clathrin heavy
chain gene (Chen and Graham, 1998), and an auxilin homo-
logue required to uncoat clathrin-coated vesicles (Gall et al.,
2000). As implicated from this screen, Drs2p function is
required for a subset of ARF and clathrin-dependent path-
ways mediating protein transport from the TGN to the cell
surface, as well as between the TGN and early endosome
(Gall et al., 2002; Saito et al., 2004; Furuta et al., 2007; Liu et al.,
2008). The Dnf P4-ATPases cannot compensate for the loss of
Drs2p in the exocytic and early endosome transport path-
ways. However, functional overlap between Drs2p and
Dnf1p is observed in the transport of proteins from the TGN
to late endosomes and the vacuole (Hua et al., 2002).
Kes1p is a negative regulator of vesicle budding in the
TGN-early endosomal system, although the mechanistic ba-
sis of this repression is not known (Mousley et al., 2007). Loss
of function kes1 alleles were recovered in screens for sup-
pressors of the kre11 and sec14 protein trafficking mutants
(Jiang et al., 1994; Fang et al., 1996). Kre11p is a component of
the TRAPPII nucleotide exchange complex for the Rab proteins
Ypt31p and Ypt32p, which regulate vesicle budding from the
TGN and early endosomes (Chen et al., 2005; Morozova et al.,
2006; Furuta et al., 2007). Sec14p is a yeast phosphatidylino-
sitol/phosphatidylcholine (PI/PC) transfer protein required
for exocytic vesicle budding from the TGN (Novick et al.,
1980; Bankaitis et al., 1990). Sec14p down-regulates PC syn-
thesis through the CDP-choline pathway (McGee et al., 1994)
and also stimulates phosphoinositide synthesis (Rivas et al.,
1999), perhaps by presenting substrate to PI 4-kinase (Schaaf
et al., 2008). Thus, Sec14p helps generate a membrane com-
position that is permissive for vesicle budding. Disruption of
KES1 can bypass the essential requirement for Sec14p by a
mechanism that is downstream of the CDP-choline pathway
for PC synthesis (Fang et al., 1996; Li et al., 2002). Kes1p is
recruited to TGN membranes by PI 4-phosphate (PI4P) and
may impart an inhibitory influence on vesicle budding by
depressing PI4P levels or competing with other effectors for
this lipid (Li et al., 2002; Fairn et al., 2007).
Here, we report that kes1? is also a suppressor of drs2
alleles, although kes1? cannot bypass the essential function
of the DRS2/DNF family of P4-ATPases. Genetically, Kes1p
seems to repress Dnf and Drs2p function at the TGN, and we
provide biochemical evidence that Kes1p potently antago-
nizes Drs2p flippase activity in purified TGN membranes.
Surprisingly, we also found that Kes1p is hyperactive in
drs2? cells and causes a significant increase in the rate of
sterol transport to intracellular membranes. Thus, Drs2p
also antagonizes the influence of Kes1p on intracellular ste-
rol transport. These observations imply that a system of
checks and balances between a P4-ATPase and oxysterol
binding protein controls vesicular and nonvesicular trans-
port processes essential for membrane biogenesis.
MATERIALS AND METHODS
Filipin, nystatin, SDS, calcofluor white (CW), and mevastatin were purchased
from Sigma-Aldrich (St. Louis, MO). Papuamide B and Ro09-0198 (Ro)-
peptide were generous gifts from Raymond Andersen (University of British
Columbia, Vancouver, BC, Canada) and Masato Umeda (Kyoto University,
Kyoto, Japan), respectively. [35S]methionine was from PerkinElmer Life and
Analytical Sciences (Boston, MA), and the yeast knockout collection was from
Invitrogen (Carlsbad, CA).
Media and Strains
Yeast strains used in this study are listed in Supplemental Table 1 and were
grown in standard rich medium (YPD) or synthetic defined (SD) minimal
media containing the required nutritional supplements (Sherman, 1991).
Yeast transformations were performed using a lithium acetate method (Gietz
and Woods, 2006). KES1 was disrupted by polymerase chain reaction (PCR)-
mediated replacement with HIS3 from Saccharomyces kluyveri by using prim-
ers forward (5?-TCG AAA AAT TTA TAA GAT TTA GTC TCA AGA ATT
TCA AGT CCG GAT CCC CGG GTT AAT TAA-3?) and reverse (5?-ATT AGT
GCA ACG GTA ACA AGT TGT TAC TTT ATC GTT CTC CGA ATT CGA
GCT CGT TTA AAC-3?) primers with the pFA6a-His3MX6 template as de-
scribed previously (Longtine et al., 1998). In other strains, KES1 was replaced
with URA3 by transformation with pRE352 (Fang et al., 1996) digested with
EcoRI and BamHI. The BY4741 kes1? drs2? strain was constructed using
pZH523 (Hua et al., 2002) to disrupt DRS2 in the BY4741 kes1? strain back-
ground. For strains used for the cholesterol transport assays, UPC2 was
replaced with upc2-1 by homologous recombination to allow these strains to
take up exogenous cholesterol during aerobic growth (Raychaudhuri et al.,
To test for growth inhibition, cells in early log phase were diluted to 0.1
OD/ml in rich media containing potential inhibitors at various concentra-
tions, and growth was monitored using an enzyme-linked immunosorbent
assay plate reader. The OD600after 36 h in rich media without inhibitor was
defined as 100% growth for a particular strain and used to normalize data
with inhibitor (? SD; n ? 6). Tests for growth at high-pressure was done as
described previously (Abe and Minegishi, 2008).
Screen for Suppressors of drs2? and Cloning of KES1
Individual colonies of MAT? and MATa drs2? strains (SEY6210 drs2?::TRP1
and SEY6211 drs2?::LEU2) were streaked on rich media and incubated at the
nonpermissive growth temperature (20 or 17°C) to select for spontaneous
bypass suppressors. Cold-resistant (CR) suppressor strains were backcrossed
to parental strains to define recessive and dominant suppressor alleles. Re-
cessive suppressor mutants were intercrossed to define two complementation
groups suppressor of drs2 knockout (SDK) 1 and SDK2. To clone SDK1, strain
BMY1001 (drs2? sdk1-1) was transformed with a genomic library (Goodson
et al., 1996) and ?50,000 Leu?transformants were replica plated and incu-
bated at 17°C for 5–7 d to screen for cold-sensitive colonies. The genomic
library plasmids pBMP1 and pBMP2 were rescued from two cold-sensitive
(cs) colonies and were identical in restriction digestion pattern. pBMP1 re-
stored cs growth to BMY1001 upon retransformation and DNA sequencing
indicated that it carried an 8.6-kbp fragment of chromosome XVI containing
Microscopy and Immunological Methods
Imaging of cells expressing green fluorescent protein (GFP) fusion proteins
and stained with filipin was done as described previously (Beh and Rine,
2004; Liu et al., 2007). Samples for electron microscopy were prepared as
described previously (Chen et al., 1999). Sections (50–100 nm) were observed
on a CM12 electron microscope (Philips, Eindhoven, The Netherlands). Ves-
icles (100 nm in diameter) were counted in 50 cell sections for each strain.
Error bars are SD (n ? 2). Metabolic labeling, immunoprecipitation (Graham,
2001), and immunoblotting (Chen et al., 1999) were performed as described
previously. An Odyssey infrared fluorescence detector (LI-COR, Lincoln, NE)
was used to quantify Western blots and Coomassie-stained gels.
Phospholipid Translocase Assays
The methods for purifying TGN membranes and assaying Drs2p- and ATP-
dependent 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-PS translocase activities
have been described previously (Natarajan and Graham, 2006). Briefly, equal
volumes of purified TGN membranes (0.5 mg/ml), NBD phospholipid (10
?M), and an ATP regenerating system (without ATP) were mixed on ice and
then shifted to 37°C to initiate the assay. After 2 h of incubation at 37°C with
no ATP, the samples were split and 3 ?l of 100 mM ATP or buffer H (10 mM
Antagonism between Drs2p and Kes1p
Vol. 20, June 15, 2009 2921
HEPES, pH 7.5, and 150 mM NaCl) was added per 100 ?l of sample. The kes1?
membrane samples were split again and received 1 ?l of Kes1p or buffer H
per 100 ?l of sample. The samples were incubated an additional 2 h at 37°C.
Aliquots of membranes were removed at 0, 2 (before ATP addition), and 4 h
of incubation and NBD-PS in the cytosolic leaflet was extracted onto fatty-acid
free BSA and quantified. The difference in the percentage of NBD-PS in the
cytosolic leaflet of wild-type (WT) TGN membranes incubated with or with-
out ATP at the 4-h time point was defined as 100% NBD-PS flippase activity
and was used to normalize data for other conditions assayed. Assays were
performed in duplicate and averages from at least three independent exper-
iments (? SD) are reported. Quantification of ergosterol in kes1? TGN mem-
branes was done as described previously (Hanover et al., 2005).
kes1 Suppresses drs2? Cold-sensitive Growth
Cells harboring knockout alleles of DRS2 exhibit a striking
cold-sensitive growth defect. Wild-type yeast can grow over
a range of temperatures from 10 to 40°C, whereas drs2?
strains can only grow over a temperature range of 21–40°C.
To explore the mechanism underlying this extreme cs
growth defect, we carried out a selection for spontaneous
extragenic suppressors that allowed growth of drs2? at 17°C.
Complementation tests indicated that the recessive drs2?
suppressor mutants fell into two complementation groups,
which we named SDK1 and SDK2. The SDK1 and SDK2
groups are composed of 12 and four recessive extragenic
bypass suppressors of drs2?, respectively. Figure 1A shows
the 30 and 17°C growth phenotypes of representative drs2?
sdk1 and drs2? sdk2 mutants relative to parental drs2? and
WT strains. Surprisingly, the drs2? sdk1 double mutant
formed larger colonies than even the WT strain at 17°C
SDK1 Was Cloned by Complementation of the
Cold-resistant Growth Phenotype of drs2? sdk1-1
A genomic library plasmid (pBMP1) was isolated that was
able to complement sdk1-1 upon retransformation of drs2?
sdk1-1 cells (Figure 1B). Among the five open reading frames
in pBMP1, KES1 was the most likely candidate for the sdk1-
complementing gene and as expected, a KES1 subclone com-
plemented the cold-resistant phenotype of drs2? sdk1-1 (Fig-
ure 1B). Several of the kes1 alleles isolated in the sec14
suppressor screen contained nonsense mutations, leading to
the expression of truncated, nonfunctional proteins (Fang
et al., 1996; Li et al., 2002). We examined the expression of
Kes1p in the drs2? sdk mutants by Western blotting and
found that seven of the 12 sdk1 alleles failed to express
full-length Kes1p, suggesting that they also carry nonsense
mutations (Supplemental Figure 1). These data indicate that
sdk1 strains carry mutations in KES1 (i.e., SDK1 and KES1
are the same gene) and that loss of function kes1 mutations
suppresses drs2? cs growth. Kes1p is one of seven oxysterol
binding protein homologues in yeast. None of the other six
members were able to restore a cs growth phenotype to
either drs2? sdk1-1 or drs2? sdk2, even when overexpressed
from multicopy plasmids (data not shown). We have not yet
succeeded in cloning SDK2; so, this report focuses on SDK1/
Mutations in three genes (CKI1, PCT1, and CPT1) encod-
ing enzymes of the CDP-choline pathway involved in phos-
phatidylcholine biosynthesis can bypass the essential re-
quirement of SEC14. However, we found that disruption of
CKI1 in the drs2? background failed to suppress cs growth
of drs2? (Figure 1C). Hence, inactivation of phosphatidyl-
choline biosynthesis via the CDP-choline pathway does not
drs2? sdk1-1 (BMY1001), and drs2? sdk2-64 (BMY1164) strains streaked onto rich medium and incubated at 30 and 17°C. (B) KES1 confers cs
growth to drs2? sdk1-1. The WT, drs2?, and drs2? sdk1-1 strains were transformed with empty plasmid (pRS315), pBMP1 (genomic library
clone carrying KES1) or a KES1 subclone (pCTY244) and 10-fold serial dilutions were spotted on minimal medium and incubated at 17°C.
(C) Serial dilutions of WT (BY4742), drs2? (ZHY615M2D), cki1? (BY4742 YLR133W), drs2? cki1? (KLY891), sac1? (BY4742 YKL212W), and
drs2? sac1? (KLY901) strains were incubated on rich medium at 20C and 17C. (D) Equal cell numbers of WT (BY4742), drs2? (ZHY615M2D),
kes1? (BMY02), and drs2? kes1? (BMY01) strains were seeded in minimal medium and incubated at atmospheric (0.1 mPa) or high pressure
(25 MPa) for 10 h at 30°C.
kes1 is a suppressor of drs2? cold-sensitive and high-pressure growth defects. (A) Growth of WT (SEY6210), drs2? (SEY6210 drs2?),
B.-P. Muthusamy et al.
Molecular Biology of the Cell2922
exert bypass suppression of drs2?. Deletion of SAC1, a phos-
phoinositide phosphatase, increases PI4P levels and sup-
presses sec14-ts (Rivas et al., 1999). SAC1 deletion suppressed
the cold sensitivity of drs2? at 20°C but not at 17°C (Figure
1C). However, the sac1? single mutant also failed to grow at
17°C and so the lack of drs2? suppression at this tempera-
ture may not be significant.
A high percentage of Saccharomyces mutants that cannot
grow at low temperature, including drs2?, also fail to grow
at high hydrostatic pressure (Abe and Minegishi, 2008). This
strong positive correlation suggests that the mutants have
a defect in membrane order and fluidity. To determine
whether kes1? would also suppress the high-pressure
growth defect of drs2? at 30°C, we measured the growth
of WT, drs2?, kes1?, and drs2? kes1? strains at atmo-
spheric pressure (0.1 MPa) and high pressure (25 MPa;
?250 kg/cm2) in liquid medium over a 10-h period. Un-
der these conditions, drs2? grew slower than WT or kes1?
cells at 0.1 MPa, and this growth defect was exacerbated
at 25 MPa. At both pressure conditions, the drs2? kes1?
cells showed improved growth, indicating that the drs2?
high-pressure sensitivity was at least partially suppressed
by kes1? (Figure 1D).
kes1? Suppression of drs2? Requires DNF P4-ATPase
Drs2p is part of an essential group of P4-ATPases that also
includes Dnf1p, Dnf2p, and Dnf3p (Hua et al., 2002). In the
absence of Dnf P4-ATPases (dnf1,2,3? cells), Drs2p can sup-
port growth of yeast over the full temperature range by
itself, but the drs2? dnf1,2,3? quadruple mutant is dead at
any temperature (Hua et al., 2002). To determine whether
kes1? could bypass the essential requirement for DRS2/DNF
function, or whether the mechanism of suppression required
the presence of Dnf ATPases, we tested whether deletion of
KES1 would rescue viability of a drs2? dnf1,2,3? strain. A
strain was generated that carries null alleles of all four
members of the DRS2/DNF group by first introducing a
wild-type copy of DRS2 on a URA3-based plasmid (pURA3-
DRS2) (Hua et al., 2002). This quadruple mutant strain (drs2?
dnf1,2,3?) could not lose pURA3-DRS2 and grow on
5-fluoro-orotic acid (5-FOA) (Figure 2A), a compound that
kills cells retaining URA3-based plasmids. In contrast, WT
and drs2? cells were able to lose the pURA3-DRS2 plasmid
and form colonies on 5-FOA plates. Importantly, the drs2?
dnf1,2,3? kes1? strain failed to grow on the 5-FOA plate
(Figure 2A). Therefore, kes1? suppression of drs2? requires
one or more Dnf P4-ATPase.
To determine which Dnf proteins are required to support
suppression, we tested the ability of kes1? to suppress dif-
ferent combinations of drs2? and dnf? alleles (Figure 2B).
Growth defects of a drs2? dnf1? strain at 30 and 17°C were
efficiently suppressed by kes1?, indicating that DNF2, DNF3,
or both can support kes1? suppression. A strain expressing
only DNF2 was weakly suppressed, whereas a strain carry-
ing only DNF1 exhibited a WT level of suppression. We
were unable to recover a drs2? dnf1,2? kes1? strain express-
ing only DNF3. However, the strain with wild-type DNF2
and DNF3 was suppressed better than the strain carrying
only DNF2, indicating some influence from DNF3. There-
fore, we conclude that all three Dnf ATPases contribute to
the suppression of drs2? by kes1?. We also tested whether
kes1? could suppress a temperature-sensitive (ts) for func-
tion allele of drs2 in the absence of the Dnf ATPases. Dis-
ruption of KES1 restored a wild-type growth rate at 37C to
a drs2-ts dnf1,2,3? strain (Figure 2C), and so loss of Kes1p
can improve the function of a crippled Drs2p.
Kes1p Attenuates Drs2p Flippase Activity at the TGN
The ability of kes1? to suppress drs2-ts in the dnf1,2,3?
background suggests that Kes1p antagonizes Drs2p activity
at the Golgi complex. To test this possibility biochemically,
TGN membranes were purified from WT, drs2?, kes1?, and
drs2? kes1? cells to assay for phospholipid translocase (flip-
pase) activity. Equal amounts of protein from each TGN
preparation were first probed for Kes1p by immunoblot to
determine whether Kes1p copurifies with these membranes
(data for WT membranes is shown in Figure 3A). Relative to
the recombinant Kes1p standard, the TGN membranes from
WT and drs2? cells carry ?2 ng (40 fmol) of Kes1p per 3 ?g
of total protein. From similar quantitative immunoblots, the
WT and kes1? membranes contain ?0.6 ng of Drs2p (4 fmol)
per 3 ?g of total protein.
Each TGN membrane preparation was assayed for
NBD-PS translocase activity as described under Materials
and Methods. The ATP-dependent translocation (flip) of
NBD-PS from the luminal leaflet to the cytosolic leaflet was
quantified and normalized to the WT membrane sample. The
kes1? membranes displayed significantly greater NBD-PS
flippase activity than WT membranes. This flippase activity
was eliminated in the drs2? membrane sample and was
substantially reduced in drs2? kes1? membranes (Figure 3B).
(A) kes1? cannot bypass the essential function of the DRS2-DNF
gene family. Strains harboring the indicated combination of null (?)
and WT (?) alleles for P4-ATPase and KES1 genes, and carrying
wild-type DRS2 on a URA3 plasmid, were spotted on minimal
media with and without 5-FOA. Strains used were BY4742,
ZHY615M2D, ZHY704, and BMY04. (B) Strains harboring the indi-
cated combination of null (?) and WT (?) alleles were spotted on
rich medium and incubated at 30 and 17°C. Strains used were
BY4742, ZHY615M2D, ZHY2149D, BMY05, ZHY708, BMY029,
ZHY7282C, and BMY039a. (C) kes1? suppresses the temperature-
sensitive growth defect of a drs2-ts dnf1,2,3? strain at 37°C. Strains
were ZHY409, ZHY410-3A, and BMY041.
Specificity for kes1? suppression of P4-ATPase mutants.
Antagonism between Drs2p and Kes1p
Vol. 20, June 15, 20092923
Thus, Drs2p was primarily responsible for the enhanced
NBD-PS flippase activity in kes1? membranes. The Dnf P4-
ATPases may have contributed the small increase in drs2?
kes1? NBD-PS flippase activity relative to drs2? membranes,
although these data are not statistically different.
It was formally possible that the enhanced NBD-PS flip-
pase activity in kes1? membranes was an indirect effect of
the chronic Kes1p deficiency. Therefore, we tested whether
adding back recombinant Kes1p to the mutant membranes
would repress the Drs2-dependent flippase activity. Addi-
tion of recombinant Kes1p to the kes1? TGN membranes
attenuated NBD-PS flippase activity to wild-type levels (Fig-
ure 3B). Remarkably, the half-maximal inhibitory concentra-
tion was in the range of 1 to 10 pg of Kes1p per 17 ?g of total
Golgi protein (0.02–0.2 pM Kes1p). For comparison, the WT
membrane preparation contained ?10 ng of endogenous
Kes1p and addition of up to 1 ?g of recombinant Kes1p to
the kes1? membranes conferred no additional inhibition of
NBD-PS flippase activity (unpublished observation). We
considered the possibility that the recombinant Kes1p was
attenuating Drs2p activity by extracting ergosterol from the
TGN membrane. However, the kes1? TGN membranes used
for these assays contained 60 ? 15 ?g of ergosterol per
milligram of protein. At 0.1 ng of Kes1p, the Kes1p to
ergosterol stoichiometry would be 1:10,000; so, it is unlikely
that this concentration of Kes1p would significantly alter the
ergosterol content of the TGN membrane.
The NBD-PS flippase activity shown in Figure 3B was
determined after 2 h of incubation with ATP, at which time
all the NBD-PS probe in the kes1? samples was flipped to the
cytosolic leaflet, and so the flippase assay was saturated. To
better assess the influence of Kes1p on the kinetics of
NBD-PS translocation, we measured the amount of NBD-PS
flipped in 15 min of incubation with ATP (Figure 3C). After
initial incorporation of the NBD-PS probe in the cytosolic
leaflet, WT and kes1? TGN membranes were incubated for
4 h without ATP to allow spontaneous redistribution of
?40% of the probe to the lumenal leaflet (NBD-PS that is
resistant to back-extraction with fatty-acid free BSA). ATP
was then added and the membranes were incubated at 37C
for 15 min before back-extraction of the probe with BSA.
Again, nearly all of the NBD-PS was flipped to the cytosolic
leaflet of the kes1? membranes in 15 min. Under these con-
per Drs2p molecule per minute in the kes1? membranes, rela-
tive to ?160 NBD-PS molecules flipped/Drs2p/min in WT
Drs2p Antagonizes the Influence of Kes1p on Sterol
Trafficking and Subcellular Distribution
Because Kes1p binds ergosterol and is implicated the intra-
cellular transport of this sterol, we examined the relation-
ship of drs2? suppression by kes1? to ergosterol metabolism
and localization. We found that drs2? cells are hypersensi-
tive to mevastatin (Figure 4A) an inhibitor of 3-hydroxy–3-
methylglutaryl-CoA reductase, the rate-limiting enzyme of
ergosterol synthesis. kes1? cells are slightly hypersensitive to
mevastatin, but the drs2? kes1? strain exhibits WT resis-
tance. Perturbation of TGN function in drs2? cells also alters
the trafficking, function, or both of cell wall biosynthetic
enzymes. This causes hypersensitivity of drs2? to CW, a
chitin-binding compound that can interfere with cell wall
assembly. By contrast to the mevastatin hypersensitivity,
kes1? does not suppress the CW hypersensitivity of drs2?
(Figure 4A). kes1? also completely suppressed the hypersen-
sitivity of drs2? to nystatin (Figure 4B), a polyene antifungal
compound that binds ergosterol in the plasma membrane.
drs2? is synthetically lethal with erg6?, a gene encoding
the ?(24)-sterol C-methyltransferase that converts zymos-
terol to fucosterol in the ergosterol biosynthetic pathway
(Kishimoto et al., 2005). Viability of a drs2? erg6? strain can
be sustained with WT DRS2 carried on a URA3 plasmid
(pURA3-DRS2). This strain cannot lose pURA3-DRS2 and
therefore failed to form colonies on media containing
5-FOA, whereas the other single and double mutants grew
well (Figure 4C). However, a drs2? erg6? kes1? strain
readily lost the pURA3-DRS2 plasmid and grew robustly on
5-FOA. Thus, kes1? suppresses the synthetic lethality be-
tween drs2? and erg6?. The data shown in Figure 4, A–C,
suggests that drs2? cells might have a defect in ergosterol
synthesis. However, a normal concentration of ergosterol in
drs2? cells has been reported previously (Fei et al., 2008), and
we have confirmed this result (data not shown).
To determine whether drs2? perturbs ergosterol subcellu-
lar distribution, we stained cells with filipin, a fluorescent
polyene antifungal compound that also binds ergosterol.
Filipin primarily stained the plasma membrane of WT cells
membranes. (A) TGN membranes were purified from WT (BY4742),
kes1? (BMY02), drs2? (ZHY615M2D), and drs2? kes1? (BMY01)
cells. The indicated amounts of WT TGN membranes and recombi-
nant Kes1p were immunoblotted to quantify Kes1p in this mem-
brane sample. (B) ATP-dependent translocation of NBD-PS from the
luminal leaflet to the cytosolic leaflet of the TGN membranes (flip-
pase activity) was measured as described under Materials and Meth-
ods. NBD-PS flippase activity in WT membranes was defined as
100% activity and used to normalize data for other samples. Error
bars are SD (n ? 3; **p ? 0.005; *p ? 0.0223 compared with kes1?
membranes using Student’s t test). (C) Kinetics of NBD-PS translo-
cation by Drs2p. Golgi membranes from WT (closed symbols) and
kes1? (open symbols) cells were incubated without ATP for 4 h to
allow passive translocation of NBD-PS into the inner leaflet. ATP
was then added and the amount of NBD-PS flipped to the outer
(cytosolic) leaflet in 15 min was measured as described under Ma-
terials and Methods.
Kes1p represses the flippase activity of Drs2p in TGN
B.-P. Muthusamy et al.
Molecular Biology of the Cell 2924
(Figure 4D), where most cellular ergosterol is localized. In
contrast, substantial intracellular filipin staining was ob-
served with drs2? cells (Figure 4D). kes1? cells also exhibited
more intracellular filipin staining than WT cells. However,
filipin primarily stained the plasma membrane of drs2?
kes1? cells (Figure 4D). In this case, we observed cosuppres-
sion because the drs2? kes1? double mutant seemed more
similar to WT than either single mutant.
Previous studies indicated that inactivation of Kes1p/
Osh4p perturbs transport of sterols from the plasma mem-
brane to the ER (Raychaudhuri et al., 2006). The toxicity of
Kes1p to drs2? and the enhanced intracellular filipin stain-
ing in this mutant suggested the possibility that Kes1p is
hyperactive in transporting sterol from the drs2? plasma
membrane to internal organelles. To test this hypothesis, we
compared the rate of esterification for exogenously supplied
radiolabeled cholesterol in WT, drs2?, kes1?, and drs2?
kes1? cells. Esterification of cholesterol requires its transport
from the plasma membrane, where it is taken up, to the ER,
where the acyl-CoA:cholesterol acyltransferases (ACATs)
are localized. S. cerevisiae will not normally take up exoge-
nous sterol in aerobic conditions. Therefore, we introduced
an altered allele of a transcription factor (upc2-1) into our
strains to permit aerobic sterol uptake (Raychaudhuri et al.,
2006). After incubation with [14C]cholesterol, the cells were
harvested at the times indicated in Figure 5, and the amount
of free cholesterol and cholesteryl ester were measured. The
drs2? cells transported cholesterol from the plasma mem-
brane to the ER 5 to 6 times faster than WT cells at 30°C, and
three times faster than WT at 20C (Figure 5, cholesteryl
ester). The drs2? cells also took up significantly more cho-
lesterol (free) than WT cells. In contrast, the drs2? kes1? cells
took up cholesterol and transported it to the ER at rates
indistinguishable from the WT cells. Therefore, Kes1p was
suppression of drs2?. (A) kes1? suppresses drs2?
hypersensitivity to mevastatin. WT (BY4742),
drs2? (ZHY615M2D), kes1? (BMY02), and
drs2? kes1? (BMY01) were incubated at 30°C
on rich media with or without mevastatin or
calcofluor white. (B) kes1? suppresses drs2?
hypersensitivity to nystatin. The same strains
were incubated in liquid rich medium with the
indicated concentration of nystatin for 36 h,
and the percentage of growth (OD600) relative
to samples without drug is shown. (C) kes1?
suppresses the synthetic lethality of drs2? with
erg6?. The indicated strains are BY4742 deriv-
atives carrying pURA3-DRS2 (PRS416-DRS2)
spotted on minimal media with or without
5-FOA and incubated at 30°C. (D) kes1? sup-
presses drs2? defects in ergosterol localization.
The same strains as in A were grown to mid-
log phase and stained with filipin as described
under Materials and Methods.
Relationship of ergosterol to kes1?
brane to ER sterol transport in drs2? cells is
Kes1p-dependent. upc2-1 derivatives of the
strains described in Figure 4A were grown at
30°C and half of the cells were shifted to 20°C
for an hour and incubated with 2 ?M [14C]cho-
lesterol Cells were removed at the indicated
times, lipids were extracted, and the amount
of cholesteryl ester and free cholesterol was
quantified as described previously (Li and
Enhanced rate of plasma mem-
Antagonism between Drs2p and Kes1p
Vol. 20, June 15, 2009 2925
required for the markedly enhanced rate of cholesterol
transport in drs2? cells.
Phospholipid asymmetry of the plasma membrane is also
perturbed in drs2? cells (Pomorski et al., 2003; Chen et al.,
2006) and we tested whether kes1? would suppress this
defect. PS and PE, which are normally restricted to the inner,
cytosolic leaflet, are aberrantly exposed on the outer leaflet
of drs2? cells. This loss of asymmetry makes drs2? hyper-
sensitive to papuamide B and Ro, antifungal compounds
that permeabilize cells exposing PS or PE, respectively. Dis-
ruption of membrane integrity can also make cells hyper-
sensitive to membrane permeating agents; so, sensitivity to
low concentrations of SDS was also examined to control for
general effects of the mutations on membrane integrity.
kes1? partially suppressed the SDS, papuamide B, and Ro
sensitivity of drs2?, but the double mutant remained signif-
icantly more sensitive to papuamide B and Ro than WT cells
(Figure 6). We conclude that loss of Kes1p improves the
plasma membrane integrity of drs2? cells, presumably by
restoring sterol content, but it does not restore the normal
asymmetric distribution of PS and PE. Surprisingly, kes1?
was also hypersensitive to papuamide B and Ro, yet re-
sistant to SDS, indicating a partial loss of membrane
asymmetry. This observation suggests that hyperactivity
of P4-ATPases may also disrupt normal plasma mem-
kes1? Does Not Suppress drs2? Defects in Protein
Trafficking between the TGN, Plasma Membrane, and
The suppression of sec14-ts secretion defects by kes1? sug-
gested that the protein trafficking defects of drs2? would
also be suppressed by kes1?. Therefore, we examined the
ability of kes1? to suppress trafficking defects in several
distinct pathways caused by drs2?. Loss of Drs2p function
perturbs formation of one class of exocytic vesicles that rely
on actin cables for efficient transport to the bud plasma
membrane (Gall et al., 2002). The sla2? mutation interferes
with actin assembly and causes an accumulation of an av-
erage of 10 post-Golgi transport vesicles per cell section in
electron micrographs. However, sla2? drs2? cells have only
an average of one to two vesicles per cell section, compara-
ble with WT cells, indicating that drs2? is epistatic to sla2?
for the vesicle accumulation phenotype and is essential for
budding these vesicles at 30°C (Figure 7A; Gall et al., 2002).
Surprisingly, kes1? did not suppress the drs2? defect in
budding in these post-Golgi exocytic vesicles because the
sla2? drs2? kes1? cells contained about the same low num-
ber of vesicles as sla2? drs2? cells (Figure 7A).
Loss of Drs2p function also perturbs trafficking of Snc1p,
an exocytic SNARE, which normally cycles in a TGN 3
plasma membrane 3 early endosome 3 TGN loop. In wild-
type cells, Snc1-GFP primarily localized to the bud plasma
membrane, whereas in drs2? Snc1-GFP was mislocalized to
internal punctate structures (Figure 7B; Hua et al., 2002),
reflecting a defect in the early endosome to TGN transport
step (Saito et al., 2004). We found no difference in the local-
ization of Snc1-GFP to intracellular punctate structures in
drs2? and drs2? kes1? cells (Figure 7B). We also found no
evidence for kes1? suppression of the drs2? defect in AP-1–
dependent trafficking of chitin synthase between the TGN
and early endosome (Supplemental Figure 2).
kes1? Suppresses drs2? Defects in TGN to Vacuole
The protein trafficking steps examined above are disrupted
at high temperatures (30–37°C) as well as low temperatures
in drs2? cells. In contrast, transport of carboxypeptidase Y
(CPY) from the Golgi to the vacuole is kinetically delayed in
drs2? cells at low temperatures but is normal at 30°C (Chen
et al., 1999). We tested whether kes1? would suppress this
cold-sensitive defect in CPY transport kinetics by using
pulse-chase analyses. CPY is initially synthesized in the ER
as a 67-kDa p1 precursor form that is further modified in the
Golgi to the 69 kDa p2 precursor. p2 CPY is then sorted from
secretory cargo at the TGN, transported to the late endo-
some and on to the vacuole where it is processed to the
61-kDa mature form (mCPY) (Stevens et al., 1982). WT,
drs2?, kes1?, and drs2? kes1? strains were labeled with
35S-amino acids and chased at 30 and 15°C for the times
indicated in Figure 8A. At 30°C, no significant difference
was found for CPY maturation kinetics in these four strains.
In contrast, drs2? cells exhibited a threefold kinetic delay in
CPY transport at 15C relative to WT and kes1? cells (Figure
8A). This cold-sensitive CPY transport defect was com-
pletely suppressed in the drs2? kes1? cells.
membrane phospholipid asymmetry. WT, drs2?, kes1?,
and drs2 ?kes1? strains (same as in Figure 3C) were
subcultured to 0.1 OD600/mlin rich media with or with-
out papuamide B, Ro090198 (Ro peptide), or SDS and
incubated at 30°C for 36 h. Percentage of growth was
determined as described in Figure 4B.
kes1? does not suppress drs2? loss of plasma
B.-P. Muthusamy et al.
Molecular Biology of the Cell2926
In addition, p2 CPY was not fully formed in drs2? cells at
15°C (p1 and p2 CPY were not completely resolved by
SDS-PAGE), indicating a partial defect in a terminal man-
nosylation event catalyzed within late Golgi compartments
(Chen et al., 1999). This cold-sensitive glycosylation defect
was also completely suppressed by kes1?. We also examined
the transport kinetics of carboxypeptidase S (CPS), another
vacuolar protein that seems to follow the same route to the
vacuole as CPY (Costaguta et al., 2001). The drs2? cells also
showed a cs, 3-fold kinetic defect in the transport of CPS to
the vacuole that was suppressed by kes1? (data not shown).
Drs2p and Dnf P4-ATPases have a redundant function in
the transport of CPY and alkaline phosphatase (ALP) to the
vacuole (Hua et al., 2002). In contrast to CPY and CPS, ALP
seems to be transported directly from the Golgi to the vac-
uole without passing through an endosomal intermediate.
ALP vacuolar transport is weakly perturbed by drs2?, but a
strong defect is observed in drs2? dnf1? double mutants. To
test for kes1? suppression of this transport defect, we exam-
ined the localization of ALP-GFP in WT, drs2? dnf1?, and
drs2? dnf1? kes1? cells. ALP-GFP was mislocalized to punc-
tate structures outside the vacuolar membrane in drs2?
dnf1? (Figure 8B, arrowheads). In contrast, ALP-GFP was
properly targeted to the drs2? dnf1? kes1? vacuolar mem-
brane (Figure 8B).
The steady-state levels of CPY and ALP precursor forms,
which are normally transient and hard to detect by Western
blot of WT cell lysates, were readily apparent in the drs2?
dnf1? cells because of the trafficking defect. The accumula-
tion of these precursors was completely suppressed in drs2?
dnf1? kes1? cells (Figure 8C), indicating that kes1? sup-
pressed the trafficking defect. The Gga1p and Gga2p clathrin
adaptors are also implicated in the CPY and CPS transport
pathway (Costaguta et al., 2001). By comparison, gga1?
gga2? showed a similar accumulation of p2 CPY as drs2?
dnf1?, but this accumulation of the precursor was not
suppressed in a gga1? gga2? kes1? strain. Thus, kes1? cells
retain their dependence on GGAs for CPY transport but
become less discriminate for the P4-ATPase requirement.
ing between the TGN, plasma membrane, and early endosome. (A)
kes1? does not suppress the drs2? defect in budding post Golgi
exocytic vesicles. WT (SEY6210), sla2? (TGY1912), drs2? sla2?
(CCY642), and drs2? sla2? kes1? (BMY15) strains were grown to
mid-log phase in rich media at 30°C and processed for electron
microscopy as described previously. Eighty- to 100-nm vesicles
were counted in 50 sections for each strain. Error bars are SD (n ?
2). (B) kes1? does not suppress the Snc1p recycling defect of drs2?
cells. WT, drs2?, kes1?, and drs2? kes1? cells (same strains as Figure
4C) expressing GFP-Snc1p were grown to mid-log phase at 30°C
and viewed by fluorescence microscopy.
kes1? does not suppress drs2? defects in protein traffick-
tein trafficking. (A) kes1? suppresses a drs2? cs defect in the kinetics
of CPY transport to the vacuole. WT, drs2?, kes1?, and drs2? kes1?
cells were grown to mid-log phase in minimal medium at 30°C, half
of the culture was shifted to 15°C for 15 min, and the cells were
labeled with [35S]methione/cysteine for 10 min. Cells were chased
with cold methionine/cysteine for the indicated times. CPY was
recovered from each sample by immunoprecipitation and subjected
to SDS-PAGE and autoradiography. (B) kes1? suppresses drs2?
dnf1? defects in ALP localization. WT (BY4742) drs2? dnf1?
(ZHY2149D), and drs2? dnf1? kes1? (BMY03) cells harboring
pGO41 (GFP-ALP) were grown at 30°C to mid-log phase and ex-
amined by fluorescence microscopy. (C) Steady-state distribution of
CPY and ALP precursor and mature forms. WT, drs2? dnf1?, drs2?
dnf1? kes1? (same strains as in B), gga1? gga2? (KLY691), and gga1?
gga2? kes1? (BMY40a) cells were grown in rich media at 30°C to
mid-log phase. Whole cell lysates were prepared and immunoblot-
ted to detect precursor (pro) and mature (m) forms of CPY and ALP.
kes1? suppresses drs2? defects in TGN to vacuole pro-
Antagonism between Drs2p and Kes1p
Vol. 20, June 15, 20092927
In this report, we provide mechanistic insight into the ex-
treme cold-sensitive growth defect of drs2? cells and un-
cover a mutually antagonistic relationship between Drs2p
and Kes1p. Genetic data indicate that Kes1p is hyperactive
in drs2? and inhibits growth of these cells at low tempera-
ture, most likely through inhibition of Dnf P4-ATPases at the
Golgi and their function in TGN-to-late endosome protein
transport. Kes1p hyperactivity in drs2? cells also causes a
substantial increase in the flux of exogenously applied sterol
from the plasma membrane to sites of esterification and an
alteration in the distribution of endogenous ergosterol.
Kes1p also represses Drs2p function as deletion of KES1 can
suppress a drs2-ts allele and causes hyperactivity of Drs2p-
dependent flippase activity in purified TGN membranes.
Remarkably, flippase activity is attenuated in vitro by addi-
tion of picomolar concentrations of recombinant Kes1p to
kes1? TGN membranes. We suggest that mutual repression
between Kes1p and Drs2p provides a critical homeostatic
mechanism for controlling the intracellular trafficking of
both protein and sterol.
The recovery of kes1? in a screen for suppressors of drs2?
cold-sensitive growth further emphasizes the importance of
Drs2p to protein trafficking in the TGN/early endosome
system and provides the first genetic link between drs2? and
sec14. In addition to kes1?, other sec14 suppressors include
mutations in genes controlling the CDP-choline pathway for
PC synthesis and SAC1, encoding a phosphoinositide phos-
phatase. Cold-sensitive growth of drs2? is not suppressed by
a CDP-choline pathway mutation, but it is partially sup-
pressed by sac1?. Therefore, the mechanism of drs2? sup-
pression by kes1? is independent of PC biosynthesis. Stt4p,
a plasma membrane PI 4-kinase, synthesizes most of the
PI4P that accumulates in sac1? mutants. Accumulation of
PI4P at non-TGN sites causes partial mislocalization of
Kes1p from the TGN, thereby relieving its repressive effect
on this organelle (Li et al., 2002). This influence on Kes1p
seems to account to the suppression of sec14-ts by sac1? and
may also explain the suppression of drs2? by sac1?.
We had suggested previously that the inability of drs2?
cells to grow at temperatures below 23°C was caused by a
failure of the Dnf P4-ATPases to support an essential Drs2p
function at the colder temperatures (Hua et al., 2002). The
current studies support this hypothesis, because suppres-
sion of drs2? growth defects by kes1? requires the presence
of Dnf P4-ATPases. Importantly, kes1? cannot suppress
drs2? defects in protein transport pathways that have a strict
requirement for Drs2p. These include the formation of one
exocytic vesicle class, and the AP-1/clathrin and Rcy1 path-
ways mediating transport between the TGN and early en-
dosomes (Gall et al., 2002; Liu et al., 2008). In contrast, drs2?
defects in ALP and CPY transport from the TGN to the
vacuole, pathways supported by either Drs2p or Dnf P4-
ATPases (Hua et al., 2002), are suppressed by kes1?. The
TGN is the likely point of suppression because Kes1p and
Drs2p normally localize to the TGN, and this is presumably
the last common site in the ALP and CPY transport path-
ways before these proteins arrive at the vacuole. Therefore,
we suggest that removal of Kes1p improves Dnf function in
the TGN so these P4-ATPases can better compensate for the
loss of Drs2p.
The ability of kes1? to restore growth of drs2? cells at low
temperature best correlates with the restoration of a wild-
type rate of CPY and CPS transport from the Golgi to the
vacuole at low temperature. The TGN 3 late endosome 3
vacuole route followed by these proteins is the only protein
trafficking pathway we have found that is normal in drs2?
cells at permissive growth temperatures (Chen et al., 1999)
and defective at nonpermissive growth temperatures. This
trafficking defect may be caused by a cs perturbation of a
GGA/clathrin pathway. The GGA and AP-1 clathrin adap-
tors seem to mediate parallel pathways emanating from the
TGN that deliver cargo to the late and early endosome,
respectively (Black and Pelham, 2000; Costaguta et al., 2001;
Hirst et al., 2001). Mutations in either pathway do not per-
turb growth of yeast; however, simultaneous loss of both
pathways severely abrogates growth (Costaguta et al., 2001;
Hirst et al., 2001). We had shown previously that drs2? cells
exhibit a constitutive loss of the AP-1 pathway and that
drs2? gga1? gga2? triple mutants are severely compromised
for growth (Liu et al., 2008). Here, we propose that the
cold-sensitive defect in growth of drs2? cells is caused by a
temperature-conditional defect in the GGA/clathrin path-
way elicited only at low temperatures, combined with a
constitutive defect in AP-1/clathrin function.
Why is Dnf function and the GGA/clathrin pathway per-
turbed at low temperature in drs2? cells, and how does loss
of Kes1p suppress this defect? Important clues came from
the analysis of ergosterol distribution and sterol trafficking
in drs2? and drs2? kes1? cells. Relative to wild type, drs2?
cells display reduced filipin staining of the plasma mem-
brane and an altered sensitivity to nystatin, but no change in
bulk ergosterol levels. These phenotypes are temperature
independent and are suppressed by kes1?, suggesting that
loss of Drs2p causes redistribution of ergosterol from the
plasma membrane to internal membranes by a Kes1p-de-
pendent mechanism. In fact, we found that drs2? cells ex-
hibit a five- to sixfold increase in the rate of exogenously
applied cholesterol esterification. This result implies an in-
creased rate of transport from the plasma membrane to the
ER, where the ACATs are localized. Elimination of Kes1p
restored a wild-type rate of esterification in spite of the
presence of six other Osh proteins in the drs2? kes1? cells.
Thus, it seems that Kes1p is the only oxysterol binding
protein homologue that is hyperactive in drs2? cells. These
data support the proposed sterol transport function for
Kes1p (Raychaudhuri et al., 2006), although we cannot rule
out the possibility that Kes1p regulates the activity of other
proteins that directly mediate nonvesicular sterol transport.
As a consequence of the altered sterol transport in drs2?, we
suggest that either an increased concentration of sterol in the
Golgi membrane, or an increased occupancy of Kes1p with
sterol, inhibits the activity of P4-ATPases in the Golgi. In the
case of drs2? cells, it is likely the combination of diminished
membrane fluidity at low temperature and the repressive
effect of Kes1p on Dnf P4-ATPases that disrupts protein
transport in the CPY/GGA pathway. The observation that
drs2? cells are high-pressure sensitive as well as cold-sensi-
tive for growth suggests a lipid packing defect may contrib-
ute to this loss of membrane fluidity.
The molecular basis for hyperactivity of Kes1p in drs2?
cells is not known. One possibility is that drs2? cells accu-
mulate a sterol that is a potent activator of Kes1p. In support
of this model, drs2? and cdc50? (noncatalytic subunit) mu-
tations exhibit a strong synthetic lethal relationship with erg
mutations (Kishimoto et al., 2005) that disrupt late steps in
ergosterol synthesis (for example erg6) and accumulate er-
gosterol precursors. The synthetic lethality between erg6?
and drs2? is completely suppressed by kes1?, indicating that
Kes1p, but not other Osh proteins, is extremely toxic to
drs2? cells producing sterols with an altered structure.
cdc50? is synthetically lethal with erg6?, erg2?, erg3?, and
erg5? (Kishimoto et al., 2005), whose products catalyze in
B.-P. Muthusamy et al.
Molecular Biology of the Cell2928
series the conversion of zymosterol to ergosta-5-7-22-24(28)-
tetraenol, the final intermediate to ergosterol. Accumulation
of one or more of the intermediates in this pathway may
stimulate the repressive activity of Kes1p at the Golgi. Con-
sistently, cells depleted for Cdc50p and Erg3p massively
accumulate Golgi and/or endosomal structures, reminiscent
of strains deficient for Drs2 and Dnf P4-ATPases (Hua et al.,
2002; Kishimoto et al., 2005).
However, it is also possible that Kes1p hyperactivity is a
function of membrane disorganization rather than its sterol
ligand. For example, Drs2p may help establish a plasma
membrane structure with high-affinity for ergosterol, and
therefore a low escape potential for this sterol, relative to the
ER or Golgi (see Maxfield and Menon, 2006 and Lange and
Steck, 2008 for discussion of cholesterol escape potential).
Consistent with this possibility, loss of plasma membrane
asymmetry in erythrocytes and fibroblasts has been shown
to increase the escape potential of cholesterol from the
plasma membrane (Lange et al., 2007). In yeast, a membrane
structure with low ergosterol escape potential may be estab-
lished during budding of exocytic vesicles from the TGN as
these vesicles are reported to have a concentration of ergos-
terol similar to the plasma membrane (300 ?g ergosterol/mg
protein; Daum et al., 1998), much higher than what we find
in TGN membranes (50–60 ?g ergosterol/mg protein). This
membrane organization may restrict the ability of Kes1p to
bind and extract sterol from exocytic vesicles or the plasma
membrane and redistribute it to internal membranes. In
addition, ergosterol biosynthetic intermediates may have a
reduced affinity for plasma membrane lipids, allowing a
more efficient extraction from the plasma membrane (Li and
Prinz, 2004; Lange and Steck, 2008). Thus, drs2? and erg
perturbations may additively increase occupancy of Kes1p
with sterol, thereby increasing its inhibitory effect on the
remaining P4-ATPases. It does not seem to be the asymmet-
ric distribution of PS and PE to the inner leaflet that is the
critical structural feature of the plasma membrane needed to
retain ergosterol. dnf1,2,3? cells exhibit a similar loss of PE
and PS asymmetry as drs2?, and yet the plasma membrane
of these cells stains normally with filipin. The neo1-ts mutant
also stains normally with filipin (unpublished observations).
Thus, Drs2p is the only P4-ATPase whose loss impacts sterol
transport or localization, suggesting a unique and undefined
influence of Drs2p on membrane organization and/or Kes1p
The role of Drs2-related P4-ATPases in establishing a
membrane structure that resists sterol extraction seems to be
conserved through evolution and is medically relevant. The
human disease progressive familial intrahepatic cholestasis
(type I) is caused by mutations in the Atp8b1 P4-ATPase
(also called FIC1) (Bull et al., 1998), which has 41% amino
acid sequence identity to Drs2p. A mouse model for Atp8b1
deficiency has been developed, and studies of these mice led
to the proposal that loss of PS asymmetry at the bile cana-
licular membrane makes this membrane more sensitive to
damage by the detergent effects of the excreted bile (Paulusma
et al., 2006; Cai et al., 2009). Normally, cholesterol excretion
into bile requires the action of the Abcg5/8 transporter, such
that an Abcg8(?/?)-deficient mouse has a very low choles-
terol output into bile. Surprisingly, mice deficient for both
Atp8b1 and Abcg8 excrete wild-type levels of cholesterol
into bile (Groen et al., 2008). The Abcg5/8-independent ex-
cretion of bile in Atp8b1-deficient mice suggests that the
disorganized structure of the bile canalicular membrane fails
to retain cholesterol leading to its nonspecific extraction into
Elimination of Kes1p also suppresses the high-tempera-
ture growth defect caused by a drs2-ts allele in the absence of
the Dnf P4-ATPases. This observation suggested that Kes1p
represses the activity of Drs2p. In support of this possibility,
we found a substantial increase in Drs2-dependent flippase
activity in TGN membranes purified from kes1? cells. Ad-
dition of recombinant Kes1p to these membranes attenuated
Drs2p activity back to wild-type levels. Remarkably, the
half-maximal inhibitory effect of Kes1 was attained at a
stoichiometry of ?1–10 Kes1p molecules per 1000 Drs2p
molecules. A 1000-fold increase in Kes1p concentration had
no further inhibitory effect on flippase activity. Thus, the
inhibitory influence of Kes1p on Drs2 activity is unlikely to
be mediated by a direct protein-protein interaction between
Kes1p and Drs2p. Consistently we have not been able to
detect a direct interaction between Drs2p and Kes1p (un-
published observations). Inhibitory concentrations of Kes1p
are also too low to significantly alter the ergosterol concen-
tration of the TGN membrane preparation. It also seems
unlikely that these low levels of Kes1p could effectively
compete with other effectors for PI4P binding, which has
been proposed to explain the negative influence of Kes1p on
vesicle budding from the TGN (Li et al., 2002).
The potency of the Kes1p attenuation of flippase activity
suggests it is acting through an enzymatic intermediate,
perhaps comparable with the signal transducing function of
oxysterol binding protein (OSBP), a mammalian homologue
of Kes1p. When bound to cholesterol, OSBP forms a complex
with protein phosphatase 2A and a tyrosine phosphatase
that attenuates signaling through the extracellular signal-
regulated kinase (ERK) pathway. In response to 25-hydroxy-
cholesterol binding, the OSBP phosphatase complex disso-
ciates and OSBP translocates onto the Golgi (Wang et al.,
2005). Thus, it is possible that Kes1p attenuates Drs2p activ-
ity by regulating protein phosphorylation at the TGN. The
Dnf P4-ATPases seem to be regulated by FPK1 and FPK2
protein kinases (Nakano et al., 2008) although it is not known
whether Drs2p is regulated by these or other protein ki-
nases. Alternatively, Kes1p may regulate the activity of PI
4-kinases or 4-phosphatases that could secondarily influence
flippase activity. Deletion of KES1 has been shown to in-
crease PI4P levels or availability at the TGN and suppresses
growth defects caused by pik1ts(Li et al., 2002; Fairn et al.,
2007). The in vitro assay for Drs2p activity in purified TGN
membranes should be amenable to testing these potential
regulatory mechanisms of Kes1p.
Drs2p plays a critical role in vesicle budding from the
TGN and early endosomes by a mechanism that is indepen-
dent of coat recruitment. We proposed previously that the
physical displacement of phospholipid from the lumenal to
cytosolic leaflet by Drs2p induces curvature in the mem-
brane that is captured and molded by coat proteins into
vesicles (Chen et al., 1999; Graham, 2004; Liu et al., 2008). It
is logical that an appropriately balanced and coordinated
activity between Drs2p and the vesicle budding machinery
would be essential for the orderly segregation of protein and
lipids into different transport pathways. Kes1p is well suited
to regulate the activity of P4-ATPases in generating mem-
brane curvature because it contains an ArfGAP lipid pack-
ing sensor (ALPS) domain that binds highly curved mem-
branes (Drin et al., 2007). The ALPS domain forms a lid over
the sterol-binding pocket of Kes1p, suggesting that lipid
packing density and/or membrane curvature may influence
sterol binding to Kes1p.
We speculate that Drs2p imparts a degree of curvature to
the TGN membrane that has an ideal set point for vesicle
formation and for establishing sterol rich raft-like membrane
Antagonism between Drs2p and Kes1p
Vol. 20, June 15, 20092929
structures for export to the plasma membrane. As Drs2p
flippase activity drives membrane curvature beyond this set
point, Kes1p would sense the stress on the membrane and
inhibit Drs2p activity until the membrane relaxes and inhi-
bition is relieved. This model is consistent the observed
mode of Kes1p inhibition of Drs2p flippase activity: Kes1p
potently prevents hyperactivity of Drs2p but will not inhibit
Drs2p activity below a basal level, even at very high con-
centrations of Kes1p. The membrane structure at this set
point would also restrict the ability of sterol transfer proteins
to extract ergosterol from the membrane. The set point
model would explain why drs2? (reduced TGN flippase
activity) and kes1? (enhanced TGN flippase activity) single
mutants both display reduced filipin staining of the plasma
membrane, whereas the double mutant seems more similar
to wild-type cells. Although many aspects of this model
remain to be tested, it provides a conceptual framework for
understanding why a system of checks and balances be-
tween a P4-ATPase and an oxysterol binding protein may
We thank Vytas Bankaitis (University of North Carolina, Chapel Hill), Chris
Beh (Simon Fraser University), and Susan Wente (Vanderbilt University) for
providing plasmids, yeast strains, and antibodies; Larry Swift and Carla
Harris for assistance with ergosterol quantitation; and Denny Kerns for
assistance with electron microscopy. We also thank Rohini Khatri, Sophie
Chen, and the other members of the Graham laboratory for support during
these experiments. This work was supported by National Institutes of Health
grant GM-62367 (to T.R.G.). This work was partly supported by the Japan
Society for the Promotion of Science (No. 18658039 to F.A.). S.R. and W.A.P.
were supported by the Intramural Research Program of the NIDDK.
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