ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 127, 61–69 doi:10.1242/jcs.153890
RESEARCH ARTICLE
Opt2 mediates the exposure of phospholipids during cellular
adaptation to altered lipid asymmetry
ABSTRACT
Plasma membrane lipid asymmetry is important for various
membrane-associated functions and is regulated by membrane
proteins termed flippases and floppases. The Rim101 pathway
senses altered lipid asymmetry in the yeast plasma membrane. The
mutant lem3D cells, in which lipid asymmetry is disturbed owing to
the inactivation of the plasma membrane flippases, showed a
severe growth defect when the Rim101 pathway was impaired.
To identify factors involved in the Rim101-pathway-dependent
adaptation to altered lipid asymmetry, we performed DNA
microarray analysis and found that Opt2 induced by the Rim101
pathway plays an important role in the adaptation to altered lipid
asymmetry. Biochemical investigation of Opt2 revealed its
localization to the plasma membrane and the Golgi, and provided
several lines of evidence for the Opt2-mediated exposure of
phospholipids. In addition, Opt2 was found to be required for the
maintenance of vacuolar morphology and polarized cell growth.
These results suggest that Opt2 is a novel factor involved in cell
homeostasis by regulating lipid asymmetry.
KEY WORDS: Lipid asymmetry, Plasma membrane, Phospholipid,
Yeast
INTRODUCTION
A common feature of the eukaryotic plasma membrane is the
difference in lipid composition between the inner (cytosolic) and
outer (extracellular) leaflets, which is called lipid asymmetry
(Devaux, 1991; Verkleij and Post, 2000). For instance,
phosphatidylserine (PtdSer) and phosphatidylethanolamine
(PtdEtn) are mostly confined to the inner leaflet, whereas
sphingolipids and phosphatidylcholine (PtdCho) are enriched in
the outer leaflet. This lipid asymmetry is generated and
maintained by ATP-dependent membrane proteins termed
flippases and floppases that mediate inward (flip) and outward
(flop) movement of lipids between the leaflets, respectively
(Axelsen and Palmgren, 1998; Hua et al., 2002; Ikeda et al., 2006;
Pomorski et al., 2003; Seigneuret and Devaux, 1984). Proper lipid
asymmetry is required for various biological processes, including
the generation of membrane potential, establishment of cell
polarity, vesicular transport, cytokinesis, blood coagulation and
removal of apoptotic cells (Chen et al., 1999; Emoto and Umeda,
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,
Japan.
*Authors for correspondence ([email protected];
[email protected])
Received 25 March 2014; Accepted 24 October 2014
2000; Fadok et al., 1992; Furuta et al., 2007; Gurtovenko and
Vattulainen, 2008; Saito et al., 2007; Toti et al., 1996). Yeast cells
lacking all known flippases are found to be inviable (Hua et al.,
2002), suggesting that lipid asymmetry is essential for cell
viability. In humans, several mutations in flippases and floppases
have been implicated in various diseases including cholestasis,
Stargardt macular dystrophy and Scott syndrome (Allikmets
et al., 1997; Bull et al., 1998; Toti et al., 1996).
Yeast cells have five phospholipid flippases of the P4-type
ATPase family (Dnf1, Dnf2, Dnf3, Drs2 and Neo1), of which
Dnf1 and Dnf2 are the main flippases in the plasma membrane
and Drs2 functions primarily in the Golgi (Hua et al., 2002). Each
flippase forms a complex with a regulatory subunit from the
Cdc50 family for its activity; for example, Dnf1 and Dnf2 require
complexation with Lem3 in order to exit from the endoplasmic
reticulum (ER) and to function as the plasma membrane flippase
(Saito et al., 2004). It was recently suggested that phospholipids
are flipped along the protein–lipid interface of P4-type ATPases
and not through their interior (Baldridge and Graham, 2012). As
for the phospholipid floppases, two members of the ABC
transporter family (Pdr5 and Yor1) are reported in yeast
(Decottignies et al., 1998). In addition, Rsb1 has been
identified in yeast as a putative floppase or translocase for
sphingoid long-chain bases (Kihara and Igarashi, 2002; Kihara
and Igarashi, 2004). It is currently unknown how these proteins
flop lipids across the bilayer.
We previously reported that the Rim101 pathway, known as an
alkaline-responsive pathway, also senses altered lipid asymmetry
caused by the deletion of LEM3 and/or PDR5 (Ikeda et al., 2008).
In this pathway, the plasma membrane protein Rim21 acts as the
sensor molecule for both altered lipid asymmetry and external
alkalization (Obara et al., 2012) and transmits the signal at the
plasma membrane (Obara and Kihara, 2014), leading to the
proteolytic activation of the transcription factor Rim101 (Peñalva
and Arst, 2004). In the alkaline response, the processed Rim101
in yeast induces transcription of alkaline-responsive genes by
suppressing the expression of transcription repressors such as
Nrg1 and Smp1, whereas in filamentous fungi the processed PacC
(a Rim101 homolog) directly induces alkaline-expressed genes
(Lamb and Mitchell, 2003; Peñalva and Arst, 2002). Several
permeases, secreted enzymes and proteins involved in
intracellular pH homeostasis are induced by changes in external
pH for adaptation (Causton et al., 2001; Lamb et al., 2001;
Peñalva and Arst, 2002; Peñalva and Arst, 2004). In contrast to
the pH response, the cellular response to altered lipid asymmetry
in the plasma membrane remains largely unknown.
In the present work, we have comprehensively analyzed the
cellular response to altered lipid asymmetry using DNA
microarray and have identified Opt2 as a novel key factor in
cellular adaptation to altered lipid asymmetry.
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Journal of Cell Science
Saori Yamauchi, Keisuke Obara*, Kenya Uchibori, Akiko Kamimura, Kaoru Azumi and Akio Kihara*
RESULTS
The Rim101 pathway is involved in adaptation to altered
lipid asymmetry
To evaluate the importance of the Rim101 pathway in adaptation
to altered lipid asymmetry, the RIM21 gene encoding the sensor
protein in this pathway was deleted in lem3D cells, in which lipid
asymmetry is disturbed. The resultant rim21D lem3D doublemutant cells suffered a severe synthetic growth defect (Fig. 1),
indicative of the involvement of the Rim101 pathway in the
response to altered lipid asymmetry. Although Rsb1, a putative
floppase for sphingoid long-chain bases, is induced by alterations
in lipid asymmetry (Kihara and Igarashi, 2002; Kihara and
Igarashi, 2004), the growth of rsb1D lem3D cells was comparable
to that of lem3D cells, indicating that Rsb1 is not important for
the adaptation.
Altered lipid asymmetry induces the expression of genes
encoding transporters and sugar-metabolizing enzymes
In order to comprehensively characterize the cellular response to
altered lipid asymmetry, DNA microarray analysis of the yeast
genome was performed on wild-type, lem3D, pdr5D (floppase
mutant) and rim21D lem3D cells. Genes induced by alterations in
lipid asymmetry (i.e. genes induced in both lem3D and pdr5D
cells) were selected; twenty-seven of these were further selected
as Rim101-pathway-dependent genes because their elevated
expression was not observed in rim21D lem3D cells
(supplementary material Table S1). These genes are expected to
encode proteins that are inducible through the Rim101 pathway
and that function in adaptation to altered lipid asymmetry, and the
list included genes encoding transporters, enzymes for glycogen
and trehalose metabolism and an arrestin-related protein.
Opt2 plays an important role in the cellular response to
altered lipid asymmetry
Among the 27 genes extracted, we focused on those encoding,
or presumably encoding, integral membrane proteins that
might function as flippases or floppases to repair altered lipid
asymmetry: MUP3, OPT2, SSU1, MAL31, YDR089W and
YJL163C. Each of these genes was then deleted in lem3D cells,
and only opt2D lem3D cells were found to exhibit a severe growth
defect similar to that of rim21D lem3D cells (Fig. 2A). Thus,
Opt2 appears to play an important role in the adaptation to altered
lipid asymmetry. Consistent with the results of DNA microarray
Fig. 1. Sensing of altered lipid asymmetry by the Rim101 pathway is
important for cell growth. SEY6210 (WT, wild type), YOK2030 (lem3D),
YOK2027 (rim21D), YYS4 (rim21D lem3D), YOK2349 (rsb1D) and YYS7
(rsb1D lem3D) cells were grown to stationary phase, serially diluted at 1:10,
spotted onto YPD plates and grown at 30˚C for 24 h.
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Journal of Cell Science (2015) 127, 61–69 doi:10.1242/jcs.153890
analysis (Fig. 2B), Opt2 levels were elevated in both lem3D and
pdr5D cells but were reduced in lem3D rim21D cells (Fig. 2C). In
addition, Opt2 levels in rim21D cells were comparable to that in
wild-type cells, suggesting that the dependency of Opt2
expression on the Rim101 pathway becomes prominent when
lipid asymmetry is altered. Deletion of OPT2 did not affect the
activation of the Rim101 pathway as monitored by proteolytic
processing of Rim101 (Fig. 2D).
To further confirm the importance of Opt2 in adaptation to
altered lipid asymmetry, Opt2 was overexpressed in rim21D
lem3D and opt2D lem3D cells from the Rim101 pathwayindependent ADH1 promoter. As expected, overexpression of
Opt2 suppressed the severe growth defect in both strains
(Fig. 2A). Given that the Rim101 pathway was originally
reported as an alkaline-responsive pathway, the involvement of
Opt2 in alkaline response was next investigated. The Rim101pathway-defective rim21D cells were found to be hypersensitive
to alkaline pH (Fig. 2E), as others have reported (Castrejon et al.,
2006). Interestingly, cells deleted for LEM3 (lem3D cells) also
exhibited similar sensitivity to alkaline pH. Deletion of OPT2 in
wild-type and lem3D cells did not affect alkaline tolerance.
Therefore, Opt2 appears to have no significant role in alkaline
response.
Opt2 cycles between the plasma membrane and the Golgi
Intracellular localization of Opt2 was monitored by using the Nterminally GFP-fused Opt2. The GFP-tagged Opt2 (GFP–Opt2)
was mainly localized to punctate structures, which were
colocalized with the late Golgi marker Sec7–mCherry, with
limited localization to the plasma membrane (Fig. 3A). When
endocytosis was blocked by transient degradation of the actin
assembly factor Las17 using the auxin-inducible degron system
(Nishimura et al., 2009; Obara and Kihara, 2014), the GFP–Opt2
signal was detected primarily in the plasma membrane (Fig. 3B).
This result indicates that Opt2 cycles between the late Golgi and
the plasma membrane by endocytosis and secretion of vesicles.
Opt2 is required for the maintenance of vacuole morphology
when lipid asymmetry is altered
Because Opt2 is implicated in the regulation of vacuolar
morphology (Aouida et al., 2009), we investigated the potential
involvement of the Rim101-pathway-dependent response to
altered lipid asymmetry in vacuolar homeostasis. In contrast to
the normal wild-type morphology in lem3D, rim21D and opt2D
cells, the vacuoles in rim21D lem3D and opt2D lem3D cells were
highly fragmented and formed small spheres that were attached to
each other (Fig. 4A). Overexpression of Opt2 from the ADH1
promoter could, however, restore the normal vacuole morphology
in both double-mutant cells. These observations suggest that Opt2
is involved in the maintenance of vacuole morphology when lipid
asymmetry is altered.
Abnormal vacuole morphology is often attributed to defects in
vesicular trafficking to the vacuole (Raymond et al., 1992); thus,
we examined two known Golgi–vacuole transport pathways [the
carboxypeptidase Y (CPY) and AP-3 pathways] in wild-type,
lem3D, opt2D and opt2D lem3D cells. A soluble vacuolar protein
such as CPY is transported from the Golgi to the vacuole through
the late endosome (the CPY pathway) (Valls et al., 1987),
whereas a vacuolar protein such as the alkaline phosphatase Pho8
is transported to the vacuole directly from the Golgi (the AP-3
pathway) (Klionsky and Emr, 1989). These two pathways were
monitored by following the processing of CPY and Pho8,
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respectively, using immunoblot analysis (Fig. 4B). In vps21D
cells in which the CPY pathway is defective, some of the Golgi
CPY (p2-CPY) was secreted to the extracellular medium as
reported in the literature (Robinson et al., 1988). By contrast, in
lem3D, opt2D, opt2D lem3D and wild-type cells, both CPY and
Pho8 were processed to their normal vacuolar forms, i.e. the
mature form of CPY (m-CPY) and the mature and soluble forms
of Pho8 (m- and s-Pho8), respectively. Next, the endocytic
pathway was monitored using the lipophilic dye FM4-64, which
is first incorporated into the plasma membrane and then
transported to the vacuole by endocytosis. Cells were treated
with FM4-64 for 15 min and chased for 10 min. In all the cells,
the FM4-64 signal was detected mainly in the vacuolar membrane
and partially at the endosome (Fig. 4C), indicating that
endocytosis is not affected in opt2D lem3D cells. Taken
together, it can be concluded that Opt2 does not play a role in
vesicular trafficking to the vacuole.
Fig. 3. Opt2 cycles between the plasma membrane and the late Golgi.
(A) YYS250 (GFP-OPT2 SEC7-mCherry) cells were grown to log phase and
subjected to fluorescence microscopy. Arrowheads indicate GFP–Opt2
(green) colocalized with Snf7–mCherry (red). (B) YOK3206 (GFP-OPT2
LAS17-HA-AID) cells were grown to log phase, treated with 500 mM 3indoleacetic acid (IAA) or ethanol (mock) for 30 min, and subjected to
fluorescence microscopy. Scale bars: 2 mm.
Opt2 is involved in the exposure of phospholipids
To gain further insight into how Opt2 mediates adaptation to
altered lipid asymmetry, we measured the degree of lipid
asymmetry in cells lacking or overexpressing Opt2 based on
their sensitivity to the toxic peptides duramycin and papuamide
B, which specifically bind to cell surface PtdEtn (Zhao et al.,
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Fig. 2. Opt2 plays an important role in adaptation to altered lipid asymmetry mediated by the Rim101 pathway. (A) SEY6210 (WT, wild type), YYS5
(lem3D), YYS12 (opt2D), YYS13 (opt2D lem3D), YOK2027 (rim21D), YYS52 (rim21D lem3D), YOK3260 (rim21D lem3D PADH-HA-OPT2) and YOK3259
(opt2D lem3D PADH-HA-OPT2) cells were grown to stationary phase, serially diluted at 1:10, spotted onto YPD plates and grown at 30˚C for 28 h. (B) DNA
microarray analysis was performed on SEY6210 (WT, wild type), YOK2030 (lem3D), YOK2368 (pdr5D) and YOK2209 (rim21D lem3D) cells. Expression
levels of OPT2 (closed circles) and, to demonstrate uniform use of poly (A)+ RNAs, ACT1 (open circles) are shown as a relative value to that in wild-type cells.
(C) Total lysates were prepared from YYS351 (OPT2-myc), YYS352 (OPT2-myc lem3D), YYS354 (OPT2-myc pdr5D), YYS355 (OPT2-myc rim21D) and
YYS356 (OPT2-myc rim21D lem3D) cells in log phase and subjected to immunoblotting with an anti-Myc antibody. Ponceau S staining was used to demonstrate
uniform protein loading. (D) SEY6210 (WT, wild type), YOK2027 (rim21D), YOK2030 (lem3D), YYS12 (opt2D), and YYS13 (opt2D lem3D) cells harboring
pFI1 (HA-RIM101) were grown to log phase and exposed to an alkaline medium (pH 8.0) for 20 min. Total lysates were then prepared and imunoblotted with an
anti-HA antibody. FL and DC denote the full-length and processed Rim101, respectively. (E) SEY6210 (WT, wild type), YYS5 (lem3D), YOK2053 (rim21D),
YYS4 (rim21D lem3D), YYS12 (opt2D) and YYS13 (opt2D lem3D) cells in stationary phase were serially diluted at 1:10, spotted onto YPD plates, and grown at
pH 8.0 and 30˚C for 91 h.
RESEARCH ARTICLE
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Fig. 4. Opt2 is involved in the maintenance of vacuole morphology
in cells with altered lipid asymmetry. (A) SEY6210 (WT, wild type),
YYS195 (lem3D), YOK2027 (rim21D), YYS52 (rim21D lem3D), YYS12
(opt2D), YYS13 (opt2D lem3D), YOK3260 (rim21D lem3D PADH-HAOPT2) and YOK3259 (opt2D lem3D PADH-HA-OPT2) cells in log phase
were stained with FM4-64 and their vacuolar morphology was examined
under a fluorescence microscope. Inset, magnified image of the region
surrounded by the dashed line. (B) Total lysates were prepared from
SEY6210 (WT, wild type), YYS5 (lem3D), YYS12 (opt2D), YYS13
(opt2D lem3D) and YOK3156 (vps21D) cells in log phase and were
subjected to immunoblotting with anti-CPY, anti-Pho8 or, to demonstrate
uniform protein loading, anti-Pgk1 antibody. Immunoblotting of each
extracellular fraction (medium) was also performed with anti-CPY
antibody. p, preform; p2, p2 Golgi form; m, mature form; s, soluble form.
(C) SEY6210 (WT, wild type), YYS5 (lem3D), YYS12 (opt2D) and
YYS13 (opt2D lem3D) cells in log phase were pulse-labeled with FM464 and examined under a fluorescence microscope. Arrowheads
indicate endosomes labeled with FM4-64. Scale bars: 2 mm.
2008) and PtdSer (Parsons et al., 2006), respectively. In lem3D
cells, PtdEtn and PtdSer, normally confined to the inner leaflet of
the plasma membrane, are exposed to the cell surface (the outer
leaflet) owing to the defective flippase activity; as a result, lem3D
cells were hypersensitive to both duramycin and papuamide B
(Fig. 5A) (Noji et al., 2006; Parsons et al., 2006).
Deletion of either OPT2 or RIM21 in lem3D cells (opt2D
lem3D or rim21D lem3D cells, respectively) conferred some
Journal of Cell Science
Fig. 5. Opt2 is involved in the exposure of PtdEtn and
PtdSer to the outer leaflet of the plasma membrane.
(A) SEY6210 (WT, wild type), YYS5 (lem3D), YYS12
(opt2D), YYS13 (opt2D lem3D), YOK2027 (rim21D),
YYS52 (rim21D lem3D), YOK3260 (rim21D lem3D PADHHA-OPT2) and YOK3259 (opt2D lem3D PADH-HA-OPT2)
cells were grown to stationary phase, serially diluted at
1:10, spotted onto YPD plates with or without 5 mM
duramycin or 0.25 mg/ml papuamide B, and grown at 30˚C
for 50 h. (B) SEY6210 (WT, wild type), YYS5 (lem3D) and
YYS13 (opt2D lem3D) cells in log phase were collected
and the surface-exposed PtdEtn was visualized using BioRo and FITC-labeled streptavidin. Arrowheads indicate
FITC signal. Scale bar: 2 mm. (C) The percentage of cells
with FITC signal is presented as the mean6s.d. (three
independent experiments); *P,0.001 (Student’s t-test).
(D) SEY6210 (WT, wild type) and YYS307 (PADH-HAOPT2) cells in log phase were harvested and subjected to
the NBD-phospholipid transfer assay. PE,
phosphatidylethanolamine; PC, phosphatidylcholine; PS,
phosphatidylserine. Values represent the mean6s.d.
(three independent experiments); *P,0.05 (Student’s
t-test).
64
tolerance to both peptides, although the growth of the double
mutants on plates without the peptides was severely retarded;
however, these cells became sensitive again when Opt2 was
overexpressed from the ADH1 promoter. Thus, Opt2 induced by
the Rim101 pathway seems to be involved in the exposure of
PtdEtn and PtdSer. However, it must be noted that deletion of
RIM21 had a greater effect than the deletion of OPT2 on the
sensitivity of lem3D cells to papuamide B. Furthermore,
overexpression of Opt2 had a milder effect on papuamide B
sensitivity in rim21D lem3D cells than in opt2D lem3D cells (see
Discussion). In rim21D cells, double deletion of DNF1 and
DNF2, both of which encode phospholipid floppases regulated by
Lem3, caused the same effects as deletion of the LEM3 gene – the
dnf1D dnf2D rim21D cells exhibited slow growth and acquired
tolerance to duramycin (supplementary material Fig. S1A),
confirming that the effect of the LEM3 deletion is indeed the
result of a defect in flip.
The surface-exposed PtdEtn was then detected by using
PtdEtn-specific biotinylated Ro09-198 (Bio-Ro) and was
visualized by using FITC-conjugated streptavidin (Iwamoto
et al., 2004) (Fig. 5B). The structure of Ro-09-198 closely
resembles that of duramycin (Noji et al., 2006). As described in
the literature (Iwamoto et al., 2004; Saito et al., 2007), PtdEtn
was highly exposed in lem3D cells but little PtdEtn was exposed
in wild-type cells. Notably, the exposed PtdEtn in lem3D cells
was significantly reduced by deletion of OPT2 (opt2D lem3D
cells), and overexpression of Opt2 was found to enhance the
surface exposure of PtdEtn in opt2D lem3D cells (Fig. 5C). We
confirmed that the levels and localization of Drs2 and Dnf3 were
comparable in these mutant cells and in wild-type cells
(supplementary material Fig. S1B,C) and thus eliminated the
possibility of increased levels of Lem3-independent flippases
causing the reduction in the amount of exposed PtdEtn in opt2D
lem3D cells. Interestingly, levels of Pdr5, the plasma membrane
floppase, were significantly elevated in opt2D lem3D cells. These
findings substantiate the involvement of Opt2 in the exposure of
PtdEtn.
The flip-flop-mediated transfer of fluorescently labeled
phospholipids (NBD–PtdEtn, NBD–PtdCho, and NBD–PtdSer)
was next assessed in cells overexpressing Opt2. After backextraction of NBD-labeled phospholipids from the outer leaflet
with bovine serum albumin (BSA), the increase in flop activity
was estimated by the decrease in the levels of NBD-phospholipids
in the inner leaflet using flow cytometry. Overexpression of Opt2
resulted in an ,25% decrease in incorporation of NBD-labeled
phospholipids into the inner leaflet (Fig. 5D), indicative of the
direct or indirect involvement of Opt2 in phospholipid flop.
Opt2 is involved in polarized cell growth
The yeast bud grows apically during the early phase of budding,
during which PtdEtn is localized to the outer leaflet at the tip of
the elongating bud. When the apical growth switches to an
isotropic growth, the exposed PtdEtn is flipped back to the inner
leaflet. This local alteration in lipid asymmetry regulates
polarized cell growth; hence, the flippase-defective lem3D cells
have an elongated morphology (Saito et al., 2007). We next
investigated whether Opt2 plays any role in apical growth by
measuring the long:short axis ratio of the cell. The ratio of lem3D
cells was significantly larger than that of wild-type cells as
predicted, whereas opt2D cells were found to have a smaller ratio
than that of wild-type cells (Fig. 6), thus being more spherical
than wild-type cells. In addition to its role in adaptation to altered
Journal of Cell Science (2015) 127, 61–69 doi:10.1242/jcs.153890
Fig. 6. Opt2 is involved in apical growth. SEY6210 (WT, wild type),
YYS12 (opt2D) and YYS5 (lem3D) cells in log phase were harvested, fixed
and stained with FITC–concanavalin-A. The ratio of the long:short axis of the
cell was calculated. For each strain, 125 individual cells were analyzed
and the value for each cell (open circles) and the mean value (white bar) are
shown. *P,0.001 (Student’s t-test).
lipid asymmetry, Opt2 appears to play an important role in apical
growth.
DISCUSSION
Opt2 plays an important role in the cellular response to
altered lipid asymmetry
We have identified Opt2, originally reported as a member of the
oligopeptide transporter family (Lubkowitz et al., 1998), as a
novel factor required for the Rim101-pathway-dependent
adaptation to altered lipid asymmetry. Although the Rim101
pathway is known as an alkaline-responsive pathway (Peñalva
and Arst, 2004), we have demonstrated that Opt2 is not involved
in the alkaline response (Fig. 2). It is also known that the OPT2
gene is not induced by external alkalization (Causton et al., 2001;
Lamb et al., 2001; Serrano et al., 2002). Interestingly, only one
putative gene (YHR214W-A) among the 27 genes extracted from
our microarray data (supplementary material Table S1) is
reported as an alkaline-responsive gene. Thus, although both
external alkalization and altered lipid asymmetry activate the
Rim101 pathway, the set of genes induced by each perturbation
is not likely to be determined solely by the Rim101 pathway,
but rather is determined in cooperation with other signaling
pathways.
Both trehalose and glycogen are known to accumulate in yeast
during stress (François and Parrou, 2001). This seems to be also
the case in response to altered lipid asymmetry, because most of
the genes encoding enzymes for trehalose and glycogen
metabolism were induced in cells with altered lipid asymmetry
(lem3D and pdr5D cells; supplementary material Fig. S2). Of
these, three genes (TPS2, GSY1 and GIP2) were induced in a
Rim101-pathway-dependent manner (supplementary material
Table S1). The ART2 and ART4 genes encoding arrestin-related
proteins were also induced by altered lipid asymmetry
(supplementary material Fig. S3) with the induction of ART2
being Rim101 pathway dependent (supplementary material Table
S1). Arrestin-related proteins are involved in the endocytic
turnover of plasma membrane proteins such as nutrient
transporters and receptors. In this regard, it is interesting that
several genes encoding transporters (or putative transporters)
were upregulated in lem3D and pdr5D cells in a Rim101pathway-dependent manner, including MUP3, OPT2, MAL31 and
SSU1 (supplementary material Table S1). It is an interesting
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subject to investigate the endocytic turnover of plasma membrane
proteins and the involvement of arrestin-related proteins in
adaptation to altered lipid asymmetry. It should be noted that the
expression of ART9 (the gene encoding the arrestin-related
protein Art9, also known as Rim8 and essential for the Rim101
pathway) was downregulated in lem3D cells and upregulated in
lem3D rim21D cells (supplementary material Fig. S3), which is
consistent with the fact that its expression is negatively regulated
by the Rim101 pathway (Lamb and Mitchell, 2003).
Opt2 is a novel type of protein involved in exposure
of phospholipids
We have provided evidence for the involvement of Opt2 in
phospholipid exposure on the extracytoplasmic leaflet. One
simple explanatory hypothesis is that Opt2 is a floppase that
directly mediates trans-bilayer movement of phospholipids. In
this case, Opt2 would represent a novel type of floppase, because
it does not belong to the ABC transporter family to which all
known floppases (except for Rsb1) belong. An alternative
possibility is that Opt2 mediates the exposure of phospholipids
indirectly, e.g. by inhibiting or activating unknown flippases or
floppases, respectively, that function during adaptation to altered
lipid asymmetry. It is also possible that Opt2 regulates the
localization of unknown flippases and floppases. Therefore, one
of the most important directions for future work would be the
establishment of an in vitro system that allows direct evaluation
of Opt2-mediated translocation of phospholipids.
Our results demonstrate that Opt2 is involved in the exposure
of phospholipids, especially in lem3D cells, in which
phospholipid flip in the plasma membrane is largely impaired.
In this regard, it is reasonable to predict that induction of Opt2
would exacerbate the phenotype of lem3D cells; however,
contrary to this assumption, we showed that induction of Opt2
is an important process in Rim101-pathway-mediated adaptation
to altered lipid asymmetry caused by LEM3 deletion. These
apparently paradoxical results might be explained follows.
Disturbance in lipid asymmetry likely affects events throughout
the cell, because lipid asymmetry is involved in a wide range of
processes including the generation of membrane potential,
vesicular trafficking, establishment of cell polarity and
cytokinesis. Therefore, the cellular response to altered lipid
asymmetry must be fairly complex. Regulated local activation or
suppression of flip-flop might be essential for a wide range of
cellular events, and several factors involved in flip-flop
(including unknown factors) are likely to be induced, activated
or repressed when lipid asymmetry is altered. Indeed, in some
cases, NBD–PtdSer is flipped more in lem3D cells than in wildtype cells (Saito et al., 2004), suggesting that some unknown
compensatory mechanism is induced in these cases. Therefore, it
is unlikely that effects caused by a reduction in flip (in lem3D
cells) can be cancelled by a simultaneous reduction in flop. Opt2
is likely to be induced as an important factor in such global
responses.
Overexpression of Opt2 completely counteracted the
duramycin resistance of lem3D rim21D cells (Fig. 5A). By
contrast, overexpression of Opt2 in wild-type cells caused a rather
small reduction in the incorporation of NBD-labeled
phospholipids into the inner leaflet of the plasma membrane
(Fig. 5D). These somewhat inconsistent results might be due to
the presence of the active plasma membrane flippases Dnf1 and
Dnf2 in wild-type but not in lem3D rim21D cells. Alternatively,
the seeming inconsistency between the results of the two assays
66
Journal of Cell Science (2015) 127, 61–69 doi:10.1242/jcs.153890
might be due to the fact that Opt2 is primarily localized to the
Golgi, with only a minor fraction located at the plasma membrane
(Fig. 3). Alternatively the differences might be due to the
dramatic difference in the incubation times used for the assays
– 20 min for the NBD-labeled phospholipid transport assay
versus 50 h for the duramycin-sensitivity assay. Thus, the results
from the NBD-labeled phospholipid assay reflect low levels of
Opt2 in the plasma membrane; by contrast, during the longer
incubation period, the Opt2-mediated lipid asymmetry generated
at the Golgi could travel to the plasma membrane through
secretory vesicles, making the cells highly sensitive to
duramycin. It could also be possible that if Opt2 functions as a
subunit of the floppase complex, the majority of overexpressed
Opt2 would not fully function because the other subunit is absent.
The effect of overexpression of Opt2 in rim21D lem3D cells
was found to be much greater on the duramycin sensitivity than
the papuamide B sensitivity, and opt2D lem3D cells showed much
weaker tolerance to papuamide B than rim21D lem3D cells
(Fig. 5A). Therefore, Opt2 might not be the sole factor that
determines the exposure of PtdSer during adaptation to altered
lipid asymmetry; instead, some other key protein(s) might be
induced by the Rim101 pathway.
Opt2 is involved in the maintenance of vacuole morphology
and polarized cell growth
We have observed vacuole fragmentation in opt2D lem3D cells,
indicative of the possible involvement of Opt2 in the maintenance
of vacuole morphology when lipid asymmetry is altered (Fig. 4).
Given that Opt2 shuttles between the Golgi and the plasma
membrane (Fig. 3), it is conceivable that the lipid asymmetry
generated by Opt2 at the Golgi and the plasma membrane might
be propagated to the vacuole membrane through the vesicular
transport pathway. The fragmentation of vacuoles could also be
the result of the improper localization and activity of proteins in
opt2D lem3D cells required for vacuole maturation, such as those
involved in the homotypic fusion of vacuoles. However, the latter
possibility is less likely because opt2D lem3D cells did not affect
the maturation of vacuolar proteins such as CPY and Pho8, which
is known to be impaired in cells defective in the homotypic fusion
of vacuoles (Wada et al., 1992). In addition, the morphology and
distribution of vacuoles appear to be different between opt2D
lem3D and homotypic fusion mutant cells – they form small
spherical vacuoles that are attached to each other (opt2D lem3D
cells) versus much smaller discrete vacuoles dispersed throughout
the cytoplasm (cells lacking the Rab-type small GTPase Ypt7
essential for the homotypic vacuole fusion) (Wada and Anraku,
1992).
We have also demonstrated that Opt2 is involved in the apical
growth of yeast cells. This finding is supported by the previous
analysis of yeast cell morphology (Ohya et al., 2005) that
revealed a smaller long:short axis ratio of opt2D cells than that of
wild-type cells at all growth stages investigated [for details, refer
to the Saccaromyces cerevisiae Morphological Database (http://
scmd.gi.k.u-tokyo.ac.jp/datamine/)]. Interestingly, the larger ratio
was reported for cells carrying a deletion of either PDR5 or
YOR1, both of which encode the phospholipid floppase. It seems
that Opt2 might be specifically involved in PtdEtn flop at the bud
tip during apical growth. Future studies should be aimed at direct
in vitro examination of the flop activity of Opt2, identification of
proteins interacting with Opt2 and elucidation of the regulation of
Opt2 function, which would greatly deepen our understanding of
cellular adaptation to altered lipid asymmetry.
Journal of Cell Science
RESEARCH ARTICLE
MATERIALS AND METHODS
Yeast culture and medium
Saccharomyces cerevisiae strains used in this study are listed in
supplementary material Table S2. Yeast cells were grown at 30 ˚C to log
phase in YPD (1% yeast extract, 2% bactopeptone and 2% D-glucose) or
synthetic complete (SC) medium (2% D-glucose and 0.67% yeast nitrogen
base without amino acids) with appropriate supplements. Alkaline
treatment was performed by adding 1 M Tris-HCl (pH 8.0) to culture
medium at a final concentration of 100 mM. A 500 mM stock solution of
3-indoleacetic acid (IAA; Nacalai Tesque, Kyoto, Japan) was prepared in
ethanol and added to the medium at a final concentration of 500 mM.
Genetic manipulation and plasmid construction
Gene disruption was performed by replacing the entire coding region of
the gene with a marker gene. Chromosome fusion of mCherry or Myc to
the C-terminus of Sec7 or Opt2, respectively, was performed using PCRbased gene disruption and modification (Longtine et al., 1998). The
sequence encoding mCherry or Myc, the ADH1 terminator and a marker
sequence was amplified by PCR from the pFA6a vector series (Longtine
et al., 1998) with a primer set specific to each gene. For the chromosomal
fusion of GFP to the N-terminus of Opt2, the sequence encoding a marker
sequence, the ADH1 promoter and the GFP tag was amplified by PCR
from the pYM-N9 (Janke et al., 2004) vector with a primer set specific to
OPT2. Amplified cassettes were inserted directly into the chromosome
by homologous recombination. Integration of the PADH1-HA-OPT2 gene
into the URA3 or TRP1 locus was performed as follows. The PADH1-HAOPT2-TOPT2 sequence was amplified by PCR from genomic DNA of
YOK3260 (PADH-HA-OPT2) to have the SacI and XhoI sites at the 59 and
39 ends, respectively. The amplified fragment was cloned into the SacIXhoI site of pRS306 or pRS304 (Sikorski and Hieter, 1989) to generate
pOK563 or pOK571, respectively. The pOK563 and pOK571 constructs
were linearized by digestion with StuI and HindIII, respectively, and
inserted at the URA3 and TRP1 loci, respectively. The plasmid for the
expression of HA–Rim101 (pFI1) was a gift from Dr. Tatsuya Maeda
(University of Tokyo, Japan).
Immunoblot analysis
Proteins were separated by SDS-PAGE and transferred to an ImmobilonTM
polyvinylidene difluoride membrane (Millipore, Billerica, MA) as
described previously (Yamagata et al., 2011). The membrane was
incubated with antibody against HA (16B12; Covance, Princeton, NJ),
CPY (Molecular Probes, Eugene, OR), GFP (598; Medical & Biological
Laboratories, Nagoya, Japan), Myc (PL-14; Medical & Biological
Laboratories), Pho8 (a gift from Prof. Yoshinori Ohsumi, Tokyo Institute
of Technology, Japan) or Pgk1 (Molecular Probes). Immunodetection was
performed using Western Lightning ECL Pro system (PerkinElmer Life
Sciences, Waltham, MA) with a bioimaging analyzer (LAS4000; Fuji
Photo Film, Tokyo, Japan) or X-ray film.
DNA microarray analysis
Total RNA was prepared from yeast cell homogenates using the RNeasy
Mini kit (Qiagen, Hilden, Germany). Poly (A)+ RNA was purified using the
mRNA Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ).
The quality of RNA was verified by electrophoresis using Agilent 2100
Bioanalyzer (Agilent Technologies, Santa Clara, CA). A total of 200 ng of
poly (A)+ RNA was labeled with Cy3 using Low Input Quick Amp
Labeling Kit (Agilent Technologies), and hybridized with the Yeast (V2)
Gene Expression Microarray (Agilent Technologies) according to the
manufacturer’s instructions. The microarray was scanned with the Agilent
G2565BA microarray scanner (Agilent Technologies), and the fluorescence
intensity for each spot was quantified using Feature Extraction software
(Agilent Technologies). RNA from four independent cultures for each
strain was subjected to DNA microarray assay and their mean values were
analyzed using Subio Platform software (Subio, Kagoshima, Japan).
Bio-Ro staining
Exposed PtdEtn was visualized using Bio-Ro as reported previously
(Iwamoto et al., 2004; Saito et al., 2007) with slight modifications. A
Journal of Cell Science (2015) 127, 61–69 doi:10.1242/jcs.153890
1-ml culture of cells in log phase was harvested and incubated in 20 ml of
YPD medium containing 80 mM Bio-Ro at 4 ˚C for 3 h. Cells were
washed once with PBS and fixed with 5% formaldehyde in PBS at room
temperature for 1 h. Cells were then washed with spheroplast buffer
(1.2 M sorbitol, 0.1 M potassium phosphate, pH 7.4) and resuspended in
100 ml of spheroplast buffer containing 100 mg/ml zymolyase 100T
(Seikagaku Kogyo, Tokyo, Japan). After addition of b-mercaptoethanol
to a final concentration of 28 mM, cells were incubated at 30 ˚C for
10 min and washed twice with spheroplast buffer. Spheroplasted cells
were attached to poly-L-lysine-coated multiwell slides and incubated in
PBS containing 0.1% BSA at room temperature for 20 min. Cells were
washed three times with PBS, and incubated in PBS containing 5 mg/ml
Fluorescein–streptavidin (Vector Laboratories, Burlingame, CA) at room
temperature for 1 h. After five washes with PBS, cells were suspended in
ProLong Gold Antifade reagent (Life Technologies, Carlsbad, CA) and
subjected to fluorescence microscopy.
NBD-phospholipid transfer assay
NBD–PtdEtn (1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]
hexanoyl}-sn-glycero-3-phosphoethanolamine), NBD–PtdCho (1-myristoyl
-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3phosphocholine) and NBD–PtdSer (1-palmitoyl-2-{6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine) were
purchased from Avanti Polar Lipids (Alabaster, AL). For each strain,
cell solutions with an OD600 of 1.0 were harvested and the cells were
resuspended in SC medium. After the addition of NBD–PtdEtn, NBD–
PtdCho or NBD–PtdSer at a final concentration of 50 mM, cells were
incubated at 30 ˚C for 20 min, washed with SSA medium (0.67% yeast
nitrogen base without amino acids, 2% sorbitol, 0.5% casamino acids,
20 mg/l tryptophan, 20 mg/l adenine sulfate, 20 mg/l uracil, 0.067%
sodium azide), and transferred to a new tube. Cells were further washed
twice with SSA medium containing 4% BSA and once with SSA medium
and resuspended in 100 ml of SSA medium. Each sample was diluted with
SSA medium at 1:100 and analyzed by flow cytometry using a FACS
Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). For each
strain, values were corrected by subtracting the background values for the
unstained cells.
Measurement of the long and short axes of yeast cells
Cells were grown in SC medium to log phase, and cell solutions with an
OD600 of 0.5 were harvested and the cells were resuspended in 400 ml of
SC medium. Formaldehyde and potassium phosphate buffer (pH 6.6)
were added at a final concentration of 3.7% and 100 mM, respectively,
and cells were incubated at 25 ˚C for 30 min to fix the cells. Cells were
collected by centrifugation and treated again with 100 mM potassium
phosphate buffer (pH 6.6) containing 4% formaldehyde at room
temperature for 45 min. The fixed cells were washed and resuspended
in 500 ml of 100 mM potassium phosphate buffer (pH 6.6), stained with
1.6 mg/ml FITC–concanavalin-A (Sigma, St Louis, MO) at room
temperature for 10 min, and observed under fluorescence microscopy.
The long and short axes of each cell were measured using Image J
software (National Institutes of Health, Bethesda, MD) and their ratio
was calculated. For each strain, 125 individual cells were analyzed.
Microscopy
Fluorescence was visualized using a fluorescence microscope
(DM5000B, Leica Microsystems, Wetzlar, Germany) equipped with
1006 HCX PL FLUOTAR NA 1.30 oil-immersion objective. Images
were acquired with a cooled CCD camera (DFC365FX, Leica
Microsystems) controlled with LAS AF software (version 2.60, Leica
Microsystems) and archived using Photoshop CS3 (Adobe; San Jose,
CA). In some cases, a linear adjustment was applied to enhance the image
contrast using the level adjustment function of Photoshop. To visualize
the vacuole, cells in log phase were stained with 1 mM FM4-64
(Molecular Probes) for 30 min, washed and resuspended in the medium,
and incubated for an additional 30 min. To monitor the progression of
endocytosis, cells in log phase were treated with 1 mM FM4-64 for
15 min, washed and resuspended in the same medium, and incubated for
67
Journal of Cell Science
RESEARCH ARTICLE
an additional 10 min. After addition of sodium azide to a final
concentration of 20 mM, cells were kept on ice until examined by
microscopy.
Acknowledgements
We are grateful to Prof. M. Umeda and Dr. U. Kato (Graduate School of
Engineering, Kyoto University) for providing the biotinylated Ro 09-0198 (Bio-Ro),
to Dr. T. Maeda (Institute of Molecular and Cellular Biosciences, University of
Tokyo) for providing the HA-Rim101 plasmid (pFI1) and to Prof. Y. Ohsumi (Tokyo
Institute of Technology) for providing the anti-Pho8 antibody. The template
plasmid for N-terminal tagging with GFP (pYM-N9) was provided by the European
S. cerevisiae Archive for Functional Analysis (Euroscarf, Germany) and the AID
system was from the National Bio-Resource Project (NBRP) of the MEXT, Japan.
We also thank Dr. T. Toyokuni for editing the manuscript.
Competing interests
The authors declare no competing interests.
Author contributions
S.Y., K.O., K.U., A.Kamimura and K.A. did the experiments. K.O. and K.A.
designed the experiments. S.Y., K.O. and A.Kihara analyzed the data. K.O. wrote
the manuscript.
Funding
This work was supported by a Grant-in-Aid for Scientific Research (C) [grant number
25440038], a Grant-in-Aid for Young Scientists (B) [grant number 23770135] to K.O.
and a Grant-in-Aid for Challenging Exploratory Research [grant number 25650059]
to A.Kihara from the Japan Society for the Promotion of Science.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.153890/-/DC1
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