Microbiology (2015), 161, 89–98
DOI 10.1099/mic.0.080481-0
Vsl1p cooperates with Fsv1p for vacuolar protein
transport and homotypic fusion in
Schizosaccharomyces pombe
Akira Hosomi,3 Yujiro Higuchi, Satoshi Yagi and Kaoru Takegawa
Correspondence
Kaoru Takegawa
[email protected]
Received 30 April 2014
Accepted 3 November 2014
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University,
6-10-1 Hakozaki, Fukuoka 812-8581, Japan
Members of the SNARE protein family participate in the docking–fusion step of several
intracellular vesicular transport events. Saccharomyces cerevisiae Vam7p was identified as a
SNARE protein that acts in vacuolar protein transport and membrane fusion. However, in
Schizosaccharomyces pombe, there have been no reports regarding the counterpart of Vam7p.
Here, we found that, although the SPCC594.06c gene has low similarity to Vam7p, the product of
SPCC594.06c has a PX domain and SNARE motif like Vam7p, and thus we designated the gene
Sch. pombe vsl1+ (Vam7-like protein 1). The vsl1D cells showed no obvious defect in vacuolar
protein transport. However, cells of the vsl1D mutant with a deletion of fsv1+, which encodes
another SNARE protein, displayed extreme defects in vacuolar protein transport and vacuolar
morphology. Vsl1p was localized to the vacuolar membrane and prevacuolar compartment, and its
PX domain was essential for proper localization. Expression of the fusion protein GFP-Vsl1p was
able to suppress ZnCl2 sensitivity and the vacuolar protein sorting defect in the fsv1D cells.
Moreover, GFP-Vsl1p was mislocalized in a pep12D mutant and in cells overexpressing fsv1+.
Importantly, overexpression of Sac. cerevisiae VAM7 could suppress the sensitivity to ZnCl2 of
vsl1D cells and the vacuolar morphology defect of vsl1Dfsv1D cells in Sch. pombe. Taken
together, these data suggest that Vsl1p and Fsv1p are required for vacuolar protein transport and
membrane fusion, and they function cooperatively with Pep12p in the same membrane-trafficking
step.
INTRODUCTION
In eukaryotic cells, newly synthesized proteins must be
correctly transported to the final destination. This protein
transport mechanism is conserved from yeast to humans
and is essential for the proper localization of each protein.
Therefore, the mechanism of protein transport is a critical research subject. Intracellular vesicular trafficking is a
primary mechanism of protein transport that involves
many protein components (van Vliet et al., 2003; Bowers &
Stevens, 2005; Tang et al., 2005; Watson & Stephens, 2005).
Vesicular trafficking is composed of two steps: vesicle
budding and membrane fusion. Genetic and biochemical
analyses have identified a large number of protein families
3Present address: Glycometabolome Team, RIKEN Advanced Science
Institute, Wako, Saitama 351-0198, Japan.
All sequence data in this article have been released and are available in
the database PomBase, including SPCC594.06c for vsl1+ (http://www.
pombase.org/).
Abbreviations: CPY, carboxypeptidase Y; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate;
PX, Phox homology; SNARE, soluble N-ethylmaleimide-sensitive factor
attachment protein receptor; TGN, trans-Golgi network.
080481 G 2015 The Authors
that have similar roles at each stage of the transport
process. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-anchored
proteins that were originally isolated from bovine brain
(Söllner et al., 1993). These key factors, which are conserved from yeast to humans, contain a coiled-coil region
that is involved in the membrane fusion step of vesicular trafficking (Hong, 2005; Toonen & Verhage, 2003;
Ungermann & Langosch, 2005). Many SNARE proteins
contain transmembrane domains in their C terminus and
all SNAREs have one or two SNARE motifs. Moreover,
SNARE proteins are classified into four different groups
(R-, Qa-, Qb-, and Qc-SNAREs), which form an extended
four-helix bundle and are sufficient for complex formation.
The combination of these three or four SNAREs is
important for specificity of membrane transport (McNew
et al., 2000; Parlati et al., 2000; Fukuda et al., 2000).
A total of 24 SNARE proteins have been identified in
Saccharomyces cerevisiae (Pelham, 1999), where they play
roles not only in protein transport but also in homotypic
vacuolar fusion (Wickner, 2002). For example, Vam3p
(Darsow et al., 1997; Wada et al., 1997), Vam7p (Sato et al.,
1998), Pep12p (Becherer et al., 1996), Vti1p (von Mollard
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89
A. Hosomi and others
et al., 1997) and Ykt6p (Kweon et al., 2003) are SNARE
proteins that function in vacuolar protein transport and
membrane fusion. The fission yeast Schizosaccharomyces
pombe is also exploited as a model organism for the study
of cell biology (Takegawa et al., 2003b). In Sch. pombe,
although the complete genome sequence has been reported
and a group of GTP-binding proteins known as Ypt
proteins, which act ‘upstream’ of SNARE proteins, have
been well studied (Armstrong, 2000), there have been few
reports regarding the SNARE proteins required for
vacuolar protein transport and biogenesis. Sch. pombe has
dozens of fragmented vacuoles inside the cells, which is
different from Sac. cerevisiae, and osmotic stress causes
transitory fusion of vacuoles. Sac. cerevisiae VPS33 encodes
a Sec1 family protein that is homologous to Sch. pombe
Vps33p. Sch. pombe vps33D cells show a severe defect in
vacuolar morphology (Iwaki et al., 2003), suggesting that
some SNARE proteins in Sch. pombe work on vacuolar
membrane fusion as well as in Sac. cerevisiae.
Previously, we identified Sch. pombe Fsv1p (fission yeast
syntaxin homologue required for vacuolar protein transport)
(Takegawa et al., 2003a). The fsv1+ gene encodes a typical
SNARE protein of 247 amino acids with one transmembrane
domain at the C terminus. A BLAST search revealed that Fsv1p
shows weak similarity to Sac. cerevisiae Syn8p and Tlg1p
(22 % identity in both cases). Fsv1p is required for vacuolar
protein transport, but not for sporulation or endocytosis.
Fsv1p is the first reported SNARE protein required for
vacuolar protein transport in Sch. pombe.
In addition, we also identified Sch. pombe Pep12p (Hosomi
et al., 2011). Sac. cerevisiae Pep12p and Vam3p are
mammalian syntaxin homologues. In Sac. cerevisiae, high
expression of PEP12 suppresses the defects in protein
transport and vacuolar morphology in the vam3D mutant,
and vice versa (Darsow et al., 1997), indicating that Sac.
cerevisiae Pep12p and Vam3p are crucial for vacuolar
transport and membrane fusion. We found that the SNARE
motif of Sch. pombe Pep12p shows similarity to that of Sac.
cerevisiae Pep12p (44 %), Vam3p (38 %) and H. sapiens
syntaxin-7 (40 %). Sch. pombe pep12D cells exhibit extreme
defects in vacuolar protein transport and vacuolar morphology, indicating that Sch. pombe Pep12p is required for not
only vacuolar protein transport, but also vacuolar membrane
fusion. Pep12p is the first reported SNARE protein required
for vacuolar formation in Sch. pombe (Hosomi et al., 2011).
Here, we discovered that the SPCC594.06c gene, designated
Sch. pombe vsl1+, has low similarity to Sac. cerevisiae
Vam7p. Based on the amino acid sequence, Sch. pombe
Vsl1p contains a Phox homology (PX) domain, which
specifically binds to phosphoinositides, in its N terminus
and a SNARE motif in its C terminus. These characteristics
are similar to Sac. cerevisiae Vam7p. Vsl1p is localized to
the vacuolar membrane and prevacuolar compartment,
which is likely dependent on its PX domain. In Sch. pombe,
although the single disruptant vsl1D did not show any
obvious phenotype, the double disruptant fsv1Dvsl1D cells
90
exhibited defects in vacuolar protein sorting and morphology like the pep12D cells, suggesting that Vsl1p together
with Fsv1p is required for Golgi-to-vacuole protein
transport and vacuolar membrane fusion.
METHODS
Strains and culture conditions. Escherichia coli XL-1 Blue
(Stratagene) was used for all cloning procedures. WT Sch. pombe
strains ARC039 (h+ leu1 ura4) and KJ100-7B (h90 leu1 ura4) were
provided by Yuko Giga-Hama (Asahi Glass) and Koichi Tanaka
(University of Tokyo, Japan). The disruptants cpy1D (h+ leu1 ura4D18 his2 ade6-M216 cpy1 : : ura4), vps34/pik3D (h+ leu1 ura4-D18 his2
ade6-M216 vps34/pik3 : : ura4), ste12D (h90 leu1 ura4-D18 ade6-M210
ste12 : : ura4), fsv1D (h+ leu1 ura4-D18 his2 ade6-M216 fsv1 : : ura4)
and pep12D (h+ leu1 ura4-D18 his2 ade6-M216 pep12 : : ura4) were
constructed as described previously (Tabuchi et al., 1997a; Takegawa
et al., 1995, 2003a; Onishi et al., 2003; Hosomi et al., 2011).
Colonies of Sch. pombe cells were streaked or spotted onto yeast
extract with supplements (YES; 30 g glucose l21, 5 g yeast extract l21,
75 mg l21 arginine hydrochloride, glutamic acid, histidine hydrochloride, lysine hydrochloride and uracil, 240 mg leucine l21, pH 5.3)
medium plates with or without 200 mM CaCl2, 0.1 mM CdCl2,
300 mM MgCl2, 5 mM LiCl, 10 mM MnCl2 or 3 mM ZnCl2, and
then incubated at 25, 30 or 37 uC for 2–6 days.
DNA cloning, gene disruption and plasmid construction. To
disrupt the endogenous Sch. pombe vsl1+ gene in the WT Sch. pombe
strain ARC039, the vsl1+ gene was replaced with the ura4+ gene. To
amplify a DNA fragment carrying the vsl1+ gene from Sch. pombe
genomic DNA by PCR, the following oligonucleotides were used: 59TACTTGTAAGTTGCACCGTTCACGAGTGCC-39 and 59-GTTTGCATATGCTTCATTAGAAAGCTCGTC-39. A 1.4 kb fragment of the
PCR product was ligated to the Promega pGEM-T vector. A 0.5 kb
EcoRI–EcoRI fragment was eliminated from the vsl1+ ORF and a
1.6 kb ura4+ DNA cassette was inserted. A linearized DNA fragment
lacking the vsl1+ gene was used to transform the WT strain, and
ura4+ transformants were selected. To confirm whether the vsl1+
gene was disrupted, ura4+ transformants were analysed by PCR to
verify the correct integration of the deletion construct.
pREP41-GFP-Vsl1 was constructed as follows. The vsl1+ ORF was
amplified by PCR with SalI and BamHI sites at the ends of DNA
fragment. The PCR product was digested with SalI and BamHI, and
introduced into the corresponding site of pTN54 derived from
pREP41 (Nakamura et al., 2001). Sac. cerevisiae VAM7 was amplified
from genomic DNA by PCR and was introduced into pART1
harbouring the constitutively strong adh1+ promoter (McLeod et al.,
1987), resulting in pART1-VAM7. pREP1-GFP-Fsv1, pREP41-GFPFsv1 and pAU-Gms1p-RFP were made as reported previously
(Takegawa et al., 2003a; Nakase et al., 2010). To construct pART1GFP-Vsl1 and pART1-GFP-Fsv1, gfp-vsl1+ and gfp-fsv1+ were
amplified by PCR using pREP41-GFP-Vsl1 and pREP41-GFP-Vsl1,
respectively, as templates and introduced into pART1.
Fluorescence microscopy. Sch. pombe cells were observed with an
Olympus BX-60 fluorescence microscope (Olympus), and images
were captured with a Sensys Cooled CCD camera using the software MetaMorph (Roper Scientific). To observe the localization of
Vsl1p, cells were grown to early exponential phase, and fixed with
glutaraldehyde and paraformaldehyde as described before (Nakamura
et al., 2001). The vacuoles of fission yeast WT and mutant cells were
labelled with FM4-64 (Molecular Probes; Vida & Emr, 1995) as
described previously (Iwaki et al., 2003).
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Microbiology 161
Sch. pombe Vsl1p in vacuolar protein transport and fusion
Pulse–chase and Western blot analyses. Pulse–chase and
immunoblot analyses of the vacuolar carboxypeptidase Y from Sch.
pombe (SpCPY) were performed as described before (Tabuchi et al.,
1997a). Antibody reactions were performed using a rabbit polyclonal
antibody against SpCPY. The CPY colony blot assay was done by
replica-plating freshly grown colonies onto nitrocellulose as reported
previously (Cheng et al., 2002). To analyse the GFP-fused WT Vsl1p
and Vsl1p(F41A), a rabbit polyclonal anti-GFP pAb-HRP-DirecT
(Medical & Biological Laboratories) was used. For a loading control, a
rabbit polyclonal antibody against Sch. pombe b-tubulin (Cosmo Bio)
was used.
RESULTS
Sch. pombe Vsl1p has low similarity to Sac.
cerevisiae Vam7p
Previously, there was no report of a Sch. pombe equivalent
for the Sac. cerevisiae Vam7p, which was likely because no
obvious orthologue could be identified. However, in this
study, we carefully searched the Sch. pombe genome
database by BLAST and found that SPCC594.06c shows
weak similarity to Sac. cerevisiae Vam7p. Vam7p contains a
PX domain in its N terminus, and correspondingly
SPCC594.06c also encodes a PX domain in its N terminus
(Fig. 1a, b). Vam7p functions in vacuolar fusion and is
targeted to the vacuolar membrane through its PX domain
(Sato et al., 1998). The gene product of SPCC594.06c has a
SNARE motif in its C terminus and shows low similarity to
Sac. cerevisiae Vam7p (26.7 % identity in 60 aa of the
SNARE motif) (Fig. 1a, c). Therefore, we designated this
gene vsl1+ (Vam7-like protein 1).
(a)
341 aa
Sp Vsl1p
26.7%
Sc Vam7p
(b)
1
1
316 aa
*
111
122
(c)
281
253
340
312
Fig. 1. Schematic diagram of homology between Sch. pombe (Sp)
Vsl1p and Sac. cerevisiae (Sc) Vam7p. (a) The black and hatched
boxes depict the PX domain and SNARE motif, respectively. The
identity of the SNARE motif between Sch. pombe Vsl1p and
Sac. cerevisiae Vam7p is shown. (b, c) Amino acid sequence
alignments of the PX domain (b) and the SNARE motif (c) in Vsl1p
(upper) and Sac. cerevisiae Vam7p (lower) are shown. The black
and grey shading indicates identical and similar amino acid
residues, respectively. The asterisk in (b) marks the amino acid
residue at which the point mutation was introduced in Vsl1p for the
analysis shown in Fig. 4(d, e).
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The vsl1D cells do not show a sorting defect of
SpCPY
In Sac. cerevisiae, Vam7p is required for vacuolar protein
transport (Sato et al., 1998). We previously reported the
isolation and characterization of a vacuolar marker protein,
carboxypeptidase Y from Sch. pombe (SpCPY) (Tabuchi
et al., 1997a). In WT cells, SpCPY is efficiently sorted to the
vacuole, and therefore is not secreted through the plasma
membrane. However, mutants defective in vacuolar sorting
deliver SpCPY not to the vacuole but to the outside of the
cells. To analyse the sorting effect of SpCPY in vsl1D cells,
we employed the SpCPY colony blot assay, which directly
tests cells for the secretion of SpCPY to the cell surface.
Like WT cells, the vsl1D cells did not show a defect in
sorting of SpCPY (Fig. 2a).
Next, we examined the processing of SpCPY in vsl1D cells
by pulse–chase analysis. During protein processing in WT
cells, SpCPY undergoes modifications that result in products with characteristic molecular masses; after a 15 min
pulse period, the endoplasmic reticulum- and Golgispecific precursor form (proCPY) and a small amount of
the vacuole-specific mature form (mCPY) were labelled,
and after a 30 min chase all SpCPY proteins were transported to the vacuole as the mature form. Identically in
vsl1D cells, there was no sorting defect of SpCPY observed
at 30 min after the pulse–chase (Fig. 2b).
The fsv1Dvsl1D cells display growth defects
Since the single mutant vsl1D did not show a defect in
SpCPY transport, we tried to create a double disruptant for
further investigation. A BLAST search using the Sch. pombe
genome database revealed that Fsv1p has low similarity to
Vsl1p (E-value 2.161025); hence, we generated a double
deletion mutant fsv1Dvsl1D.
First, we performed growth tests of the fsv1Dvsl1D cells
because Sch. pombe deletion mutants lacking pep12+ and
vps33+, which are genes related to vacuolar protein
transport, show growth defects under normal culture
conditions, as well as sensitivity to temperature, CaCl2
and CdCl2 (Iwaki et al., 2003; Hosomi et al., 2011). As
expected, neither of the single mutants (fsv1D or vsl1D)
showed growth defects compared with the WT cells (Fig.
2c). However, the fsv1Dvsl1D cells exhibited temperaturesensitive growth at 30 and 37 uC as well as sensitivity to
CaCl2 and CdCl2 at 25 uC, suggesting that Vsl1p and Fsv1p
have functions similar to those of Pep12p and Vps33p
(Fig. 2c).
The fsv1Dvsl1D cells exhibit severe defects in
vacuolar protein sorting and morphology
Pep12p is a syntaxin homologue and class C Vps protein,
and Vps33p is a Sec1/Munc18 family protein. These
proteins are required not only for vacuolar formation but
also for vacuolar protein transport and ion homeostasis
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A. Hosomi and others
(a)
(b)
WT
vps34Δ
cpy1Δ
vsl1Δ
WT
vsl1Δ
0
30
30
0
Chase (min)
proCPY
mCPY
(c)
YES 25°C
fsv1Δ
vsl1Δ
vsl1Δ
fsv1Δ
WT
200 mM CaCl2 25°C
YES 30°C
0.1 mM CdCl2 25°C
YES 37°C
(Iwaki et al., 2003; Hosomi et al., 2011). Therefore, we
investigated vacuolar protein sorting in fsv1Dvsl1D cells by
the CPY colony blot assay. The fsv1Dvsl1D cells showed
stronger secretion of SpCPY than fsv1D cells (Fig. 3a). This
result suggests that Vsl1p is required for vacuolar protein
transport and functions together with Fsv1p.
The Sac. cerevisiae Vam7p protein is required for vacuolar
membrane fusion in vitro and in vivo (Sato et al., 1998;
Ungermann & Wickner, 1998; Ungermann et al., 1999).
Under normal conditions, Sch. pombe has a large number
of small vacuoles, and hypotonic stress causes transitory
fusion of vacuoles (Bone et al., 1998). To examine vacuolar
morphology, WT and vsl1D cells were grown in YES
medium, stained with FM4-64 and shifted to water to
clearly observe the shape of vacuoles. The vsl1D cells had
fragmented vacuoles and hypotonic stress caused fusion of
vacuoles without any obvious difference from WT cells
(Fig. 3b).
Then we observed vacuolar morphology in the fsv1Dvsl1D
cells and found that the double mutant cells lacked any
structures resembling the vacuoles seen in WT cells (Fig.
3b). As mentioned above, neither single deletion mutant
92
Fig. 2. The vsl1Dfsv1D cells show growth
defects. (a) Immunoblot analysis of SpCPY.
Cells were grown on a nitrocellulose filter at
30 6C for 3 days and the filter was processed
for immunoblotting using a rabbit polyclonal
antibody against SpCPY. vps34D and cpy1D
cells were used as positive and negative
controls, respectively, for SpCPY missorting.
(b) Pulse–chase analysis monitoring the processing of SpCPY in vitro. WT and vsl1D cells
were pulse-labelled with Express-35S-label for
15 min at 28 6C and chased for 30 min. The
immunoprecipitated samples were separated
by SDS-PAGE on a 10 % polyacrylamide gel.
Autoradiogram of the fixed and dried gel is
shown. The positions of proCPY (110 kDa)
and mature CPY (mCPY; 32 kDa) are indicated. (c) WT, vsl1Dfsv1D, vsl1D and fsv1D
cells were incubated on YES plates with or
without 200 mM CaCl2 or 0.1 mM CdCl2 for
3 days at 25, 30 or 37 6C.
(vsl1D or fsv1D) showed defects in vacuolar morphology
and size (Fig. 3b, c) (Takegawa et al., 2003a). The vacuolar
morphology of the fsv1Dvsl1D cells is quite similar to that
of pep12D and vps33D cells. These results indicate that
Vsl1p and Fsv1p are required for vacuolar membrane
fusion and they function cooperatively in the same step of
membrane trafficking.
Vsl1p is localized to the vacuolar membrane and
prevacuolar compartment
In Sac. cerevisiae, Vam7p is localized to the vacuolar
membrane (Sato et al., 1998). To determine the localization of Vsl1p, we prepared a fusion protein construct,
GFP-Vsl1p. First, we expressed GFP-Vsl1p in the
fsv1Dvsl1D mutant and found that the fusion protein
complemented the vacuolar morphology defect (data not
shown), indicating that this GFP-fusion protein is
functional. Subsequent observation revealed that Vsl1p is
localized to the vacuolar membrane in WT cells (Fig. 4a).
Moreover, FM4-64 fluorescence was localized to punctate
structures and was partially co-localized with GFP-Vsl1p
fluorescence (Fig. 4b). To further analyse the localization of
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Microbiology 161
Sch. pombe Vsl1p in vacuolar protein transport and fusion
fsv1D
(a)
vps34D
WT
cpy1D
vsl1D
fsv1D
fsv1D
(b)
WT
vsl1D
vsl1D
fsv1D
FM4-64
H2O
(c) 40
35
30
25
20
15
10
5
0
Vacuoles (%)
Vacuoles (%)
25
20
15
10
5
Vacuole diameter (mm)
0. 0.2
2–
0. 0.4
4–
0. 0.6
6–
0. 0.8
8–
1. 1.0
0–
1. 1.2
2–
1. 1.4
4–
1. 1.6
6–
1. 1.8
8–
2. 2.0
0–
2. 2.2
2–
2. 2.4
4–
2. 2.6
6–
2. 2.8
8–
3.
0
>
3.
0
0–
0–
0. 0.2
2–
0. 0.4
4–
0. 0.6
6–
0. 0.8
8–
1. 1.0
0–
1. 1.2
2–
1. 1.4
4–
1. 1.6
6–
1. 1.8
8–
2. 2.0
0–
2. 2.2
2–
2. 2.4
4–
2. 2.6
6–
2. 2.8
8–
3.
0
>
3.
0
0
Vacuole diameter (mm)
Fig. 3. The vsl1Dfsv1D cells display severe defects in vacuolar protein transport and aberrant vacuolar morphology. (a)
Immunoblot analysis of SpCPY is shown. Cells were grown on a nitrocellulose filter at 30 6C for 3 days and the filter was
processed for immunoblotting using a rabbit polyclonal antibody against SpCPY. vps34D and cpy1D cells were used as positive
and negative controls, respectively, for SpCPY missorting. The vsl1Dfsv1D cells showed a stronger phenotype than the fsv1D
cells. (b) WT, vsl1D, vsl1Dfsv1D and fsv1D cells were grown in YES at 30 6C and stained with FM4-64. Cells were shifted to
water for 3 h and then visualized by fluorescence microscopy. (c) Quantitative analyses of vacuolar diameter in WT (white bars),
vsl1D (grey bars) and fsv1D (black bars) cells were performed with FM4-64 (left) and shifted to water for 3 h (right).
Vsl1p, we co-expressed GFP-Vsl1p and RFP-fused Gms1p,
which is a UDP-Gal transporter and is used as a Golgi
marker protein (Tabuchi et al., 1997b; Nakase et al., 2010),
and revealed that GFP-Vsl1p was not co-localized with
Gms1p-RFP (Fig. 4c). These results demonstrate that Vsl1p
is localized to the vacuolar membrane and prevacuolar
compartment.
The PX domain of Sac. cerevisiae Vam7p and phosphatidylinositol 3-phosphate [PtdIns(3)P] but not phosphatidylinositol
3,5-bisphosphate [PtdIns(3,5)P2] are necessary for the association of Vam7p with the vacuolar membrane (Cheever et al.,
2001; Boeddinghaus et al., 2002; Lee et al., 2006). In Sch.
pombe, PtdIns(3)P is provided by the PtdIns-3 kinase Vps34p
and no PtdIns(3)P is detected in vps34D cells (Takegawa et al.,
1995). To determine whether the membrane association of
Vsl1p depends on PtdIns(3)P, GFP-Vsl1p was expressed in
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Sch. pombe vps34D cells. In the mutant cells, GFP-Vsl1p was
not associated with either the vacuolar membrane or the
prevacuolar compartment (Fig. 4a). Sch. pombe ste12+ encodes
a PtdIns(3)P 5-kinase, and no PtdIns(3,5)P2 was detected in
ste12D cells (McEwen et al., 1999; Morishita et al., 2002). We
expressed GFP-Vsl1p in ste12D cells and found that GFP-Vsl1p
was localized to the vacuolar membrane and prevacuolar
compartment (Fig. 4a). In Sac. cerevisiae, a mutated Vam7p
(Y42A) fused to GFP [GFP-Vam7p(Y42A)] does not associate
with the vacuolar membrane or complement the vacuolar
protein transport defect (Sato et al., 1998; Cheever et al., 2001).
The 41st residue in the PX domain of Vsl1p is a phenylalanine
(F41), which is equivalent to the tyrosine at position 42 (Y42)
in Sac. cerevisiae Vam7p (Fig. 1b, asterisk). We constructed a
mutated fusion protein GFP-Vsl1p(F41A) and found that
the protein did not associate with the vacuolar membrane
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A. Hosomi and others
Nomarski
GFP-Vsl1
Gms1-RFP
(e)
kD
75
50
GFP-Vsl1
(F41A)
FM4-64
sl1
FP
(F -V
41 s
A) l1
(d)
GFP-Vsl1
-V
(c)
Nomarski
ste12Δ
50
FP
(b)
vps34Δ
G
WT
GFP-Vsl1
G
(a)
α-GFP
α-β-Tubulin
Fig. 4. Intracellular localization of Vsl1p. (a) Cells of WT, vps34D
and ste12D strains expressing pREP41-GFP-Vsl1p were cultured
in minimal medium for 18 h and then visualized by fluorescence
microscopy. GFP-Vsl1p was localized to the vacuolar membrane
and prevacuolar compartment in WT and ste12D cells, but was
dispersed to the cytoplasm in vps34D cells. (b) Cells of the strain
expressing pREP41-GFP-Vsl1p were cultured in minimal medium
for 18 h, stained with FM4-64 at 20 6C for 20 min and then
visualized using a fluorescence microscope with Nomarski optics.
GFP-Vsl1p localized to the FM4-64-positive structures (arrows).
(c) Gms1p-RFP-expressing construct was introduced into the
cells expressing pREP41-GFP-Vsl1p. Gms1p was used as a
Golgi marker protein. The resultant cells were cultured in minimal
medium for 18 h and visualized using a fluorescence microscope.
Note that no co-localization was observed between GFP-Vsl1p
and Gms1p-RFP. (d) Cells of the strain expressing pREP41-GFPVsl1p(F41A) were cultured in minimal medium for 18 h and then
visualized using a fluorescence microscope with Nomarski optics.
Note that GFP-Vsl1p(F41A) fluorescence was dispersed in the
cytoplasm, unlike GFP-Vsl1p. (e) Cell lysates of strains expressing
GFP-Vsl1p or GFP-Vsl1p(F41A) were analysed by Western blot
assay using an anti-GFP antibody. For a loading control, b-tubulin
was used. Note that GFP-Vsl1p(F41A) was stably expressed as
much as GFP-Vsl1p.
94
(Fig. 4d). Western blot analysis using an anti-GFP antibody
confirmed that the GFP-fusion protein of Vsl1p(F41A) was
expressed as stably as that of WT Vsl1p (Fig. 4e). These results
suggest that the localization of Vsl1p is due to its PX domain
and PtdIns(3)P but not PtdIns(3,5)P2.
vsl1+ is able to compensate for the phenotypes of
fsv1D cells
To analyse the functional relationship between Vsl1p and
Fsv1p, we searched for other phenotypes in the vsl1D and
fsv1D cells. For this reason, we investigated the growth of
these cells under a variety of culture conditions. WT, vsl1D
and fsv1D cells had no obvious growth defects when
cultured with 100 mM CaCl2, 300 mM MgCl2, 5 mM LiCl
or 10 mM MnCl2 (date not shown). However, the vsl1D
and fsv1D cells were sensitive to ZnCl2 (Fig. 5a). Next, to
check whether one of the gene products fused to GFP can
suppress the sensitivity to ZnCl2 in the mutant lacking
the other gene, we expressed GFP-Vsl1p and GFP-Fsv1p
in vsl1D and fsv1D cells. Although the expression of GFPFsv1p could not suppress the phenotype of the vsl1D cells,
GFP-Vsl1p was able to partially suppress that of the fsv1D
cells (Fig. 5b, c). To further investigate the functional
correlation of these proteins, we performed CPY colony
blot assays. In synthetic minimal medium, the fsv1D cells
secreted SpCPY. However, the fsv1D cells expressing either
GFP-Fsv1p or GFP-Vsl1p did not secrete SpCPY (Fig. 5d).
These data indicate that Vsl1p can partially replace Fsv1p.
The localization of Vsl1p depends on Pep12p and
Fsv1p
To further analyse the functional interaction among
Vsl1p, Fsv1p and Pep12p, we examined the localization
of GFP-Vsl1p and GFP-Fsv1p in Sch. pombe pep12D cells.
GFP-Vsl1p was mislocalized in both pep12D- and fsv1+overexpressing cells (Fig. 6a). Likewise, GFP-Fsv1p was
mislocalized in the pep12D cells (Fig. 6b; Takegawa et al.,
2003a), although the fusion protein was properly localized
to Golgi and prevacuolar compartments in WT cells (Fig.
6c). These results suggest that Pep12p has functions that
are closely related to those of Vsl1p and Fsv1p, which can
result in competition for appropriate localization.
Sac. cerevisiae Vam7p can partially suppress the
phenotype of Sch. pombe vsl1D cells
Finally, to confirm the functional relationship between Vsl1p
and Sac. cerevisiae Vam7p, we introduced Sac. cerevisiae
VAM7 into the Sch. pombe vsl1D cells. In fact, overexpression
of Vam7p could partially suppress the sensitivity to ZnCl2 in
vsl1D cells (Fig. 7a). Moreover, we transformed the overexpression construct of Sac. cerevisiae VAM7 into the Sch.
pombe vsl1Dfsv1D cells, then performed the FM4-64 analysis.
We confirmed that overexpression of Sac. cerevisiae VAM7
could suppress the morphology defect of vacuoles in
vsl1Dfsv1D cells (Fig. 7b; also see Fig. 3b for vsl1Dfsv1D
cells). Collectively, these results suggest that Sac. cerevisiae
Vam7p and Sch. pombe Vsl1p have similar functions.
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Microbiology 161
Sch. pombe Vsl1p in vacuolar protein transport and fusion
(a)
YES
3 mM ZnCl2
(a)
WT
(b)
WT
pep12Δ
pREP1-fsv1+
WT
vsl1D
fsvlD
(b)
GFP-Vsl1
WT
pep12Δ
vsl1D
GFP-Vsl1
GFP-Fsv1
(c)
GFP-Fsv1
WT
(c)
GFP-Fsv1
Gms1-RFP
fsv1D
GFP-Fsv1
GFP-Vsl1
(d)
WT
vps34D
cpy1D
fsv1D
fsv1D
GFP-Fsv1
fsv1D
GFP-Vsl1
Fig. 5. Vsl1p partially suppresses phenotypes of fsv1D cells. (a)
WT, vsl1D and fsv1D cells were incubated on YES plates with or
without 3 mM ZnCl2 for 2 days at 30 6C. (b) Cells of WT and vsl1D
strains transformed with pART1, pART1-GFP-Vsl1p or pART1GFP-Fsv1p were cultured in minimal medium and incubated on YES
plates containing 3 mM ZnCl2 for 6 days at 30 6C. (c) Cells of WT
and fsv1D strains transformed with pART1, pART1-GFP-Fsv1p or
pART1-GFP-Vsl1p were cultured in minimal medium and incubated
on YES plates containing 3 mM ZnCl2 for 6 days at 30 6C. Note
that GFP-Vsl1p partially suppressed the phenotype of fsv1D cells.
(d) Immunoblot analysis of SpCPY. Cells were grown on a
nitrocellulose filter at 30 6C for 3 days and the filter was processed
for immunoblotting using a rabbit polyclonal antibody against
SpCPY. vps34D and cpy1D cells were used as positive and
negative controls, respectively, for SpCPY missorting. Expression of
either GFP-Fsv1p or GFP-Vsl1p could complement or suppress,
respectively, the phenotype of fsv1D cells.
Fig. 6. Localization of Vsl1p is dependent on Pep12p and Fsv1p.
(a) pREP41-GFP-Vsl1p was introduced into WT and pep12D
strains and a strain expressing pREP1-Fsv1p. These strains were
cultured in minimal medium for 18 h and then observed by
fluorescence microscopy. GFP-Vsl1p was not properly localized in
pep12D and Fsv1p-overexpressing cells. (b) Cells of WT and
pep12D strains transformed with plasmid pREP41-GFP-Fsv1p
were cultured in minimal medium for 18 h and then observed by
fluorescence microscopy. GFP-Fsv1p was not properly localized in
pep12D cells. (c) The expressing construct of Gms1p-RFP was
introduced into cells with pREP41-GFP-Fsv1p. The resultant cells
were cultured in minimal medium for 18 h and then observed by
fluorescence microscopy. GFP-Fsv1p was partially co-localized
with Gms1p-RFP (arrows).
DISCUSSION
the fsv1D cells, suggesting that Vsl1p is needed for Golgito-vacuole protein transport, which is also supported by
Fsv1p. Additionally, the fsv1Dvsl1D cells also exhibited
an extreme defect in vacuolar morphology, indicating
that Vsl1p and Fsv1p are required for vacuolar membrane fusion. These results demonstrate that Vsl1p is an
important protein for vacuolar protein transport and the
formation of vacuoles.
In this report, we identified the novel SNARE protein
Vsl1p, which is required for vacuolar transport and
formation in Sch. pombe. Although the single vsl1D mutant
did not have a vacuolar protein sorting defect, the double
deletion fsv1Dvsl1D cells displayed a stronger defect than
The fusion protein GFP-Vsl1p was localized to the vacuolar
membrane and prevacuolar compartment in WT and
ste12D cells but not in vps34D cells. The mutated fusion
protein GFP-Vsl1p(F41A), the mutation of which was
introduced into the PX domain of Vsl1p, was localized to
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95
A. Hosomi and others
(a)
YES
3 mM ZnCl2
WT pART1
vsl1D pART1
vsl1D pART1-VAM7
(b)
FM4-64
H2O
Fig. 7. Overexpression of Sac. cerevisiae Vam7p suppresses
phenotypes of vsl1D cells. (a) Sac. cerevisiae VAM7 was cloned
into pART1, and the resultant plasmids were introduced into vsl1D
cells. WT and vsl1D cells carrying the empty vector pART1 were
used as positive and negative controls, respectively. VAM7
expressed from pART1-VAM7 in the vsl1D background partially
suppressed the sensitivity of vsl1D cells to 3 mM ZnCl2. (b) The
vsl1Dfsv1D cells expressing pART1-VAM7 were grown in YES at
30 6C, stained with FM4-64 and then visualized by fluorescence
microscopy. Cells were also shifted to water for 3 h. Note that the
vacuolar morphology defect was suppressed by expressing VAM7
compared with the host vsl1Dfsv1D cells (see Fig. 3b).
neither the vacuolar membrane nor the prevacuolar
compartment. These results suggest that PtdIns(3)P, which
is mainly provided by Vps34p, predominantly influences
Vsl1p localization through its PX domain. In Sac.
cerevisiae, Vam7p is also localized to the vacuolar
membrane, and its PX domain and PtdIns(3)P are
important for proper localization (Cheever et al., 2001).
In Sch. pombe, it is also known that PX domain-containing
proteins Vps5p and Vps17p are involved in vacuolar
protein transport (Koga et al., 2004; Iwaki et al., 2006).
Taken together, these facts seem to indicate that Vsl1p
corresponds to Sac. cerevisiae Vam7p, although the
primary structures between these proteins are not highly
conserved, except for the N-terminal PX domain and the
C-terminal SNARE motif.
We demonstrated that overexpression of Sac. cerevisiae
VAM7 could, to some extent, suppress the sensitivity to
ZnCl2 in vsl1D cells. In plant cells, it was speculated that a
vacuolar membrane transporter AtMTP1 would function
in maintaining the subcellular zinc concentration for the
ion homeostasis, suggesting that vacuoles are important
organelles for tolerance to high zinc exposure (Kobae et al.,
2004). In fact, in budding yeast, mutants in vacuolar
organization are sensitive to high zinc concentration
(Pagani et al., 2007). Based on this idea, although no
obvious phenotype on vacuolar morphology was observed,
possible perturbation in vacuolar function would induce
the zinc sensitivity in single disruptants vsl1D and fsv1D.
96
Higher expression of GFP-Vsl1p partially suppressed the
defect in vacuolar protein transport and the sensitivity to
ZnCl2 in the fsv1D cells, but GFP-Fsv1p was unable to
suppress these phenotypes in the vsl1D cells. These results
indicate that Vsl1p can replace certain functions of Fsv1p,
but Fsv1p cannot replace Vsl1p, likely because Vsl1p has a
PX domain, whereas Fsv1p does not (Fig. 8a). Previously,
we reported that Fsv1p is localized to the prevacuolar
compartment and Golgi membrane (Takegawa et al.,
2003a) and Pep12p to the prevacuolar compartment and
vacuolar membrane (Hosomi et al., 2011). In this report,
we further revealed that Vsl1p is localized to the
prevacuolar compartment and vacuolar membrane like
Pep12p. Moreover, we demonstrated that the localization
of both Vsl1p and Fsv1p was affected in pep12D cells, and
higher expression of Fsv1p caused the mislocalization of
Vsl1p. In addition, both Vsl1p and Fsv1p are categorized as
Qc-SNARE proteins (Gupta & Heath, 2002). These facts
strongly suggest that both Vsl1p and Fsv1p are partners of
Pep12p, and the functions of Vsl1p and Fsv1p are partially
overlapping (Fig. 8b). Indeed, vacuolar morphology in the
fsv1Dvsl1D cells was abnormal and quite similar to that in
the pep12D cells (Hosomi et al., 2011), although vacuolar
morphology in the single deletion mutants vsl1D and fsv1D
cells was normal. Taken together, these facts lead to the
conclusion that Vsl1p is involved in vesicle transport from
the prevacuolar compartment to the vacuole, and from the
trans-Golgi network (TGN) to the late endosome in the
absence of Fsv1p. On the contrary, Fsv1p is involved in
vesicle transport from the TGN to the prevacuolar
compartment, as well as from the late endosome to the
vacuole in the absence of Vsl1p (Fig. 8b).
Although SNARE proteins are crucial in the vesicular
membrane-trafficking pathway of both Sch. pombe and Sac.
cerevisiae, there are several differences in the mechanisms
of protein transport and membrane fusion from the TGN
to the vacuole between these yeasts. Sch. pombe Fsv1p
mainly acts from the TGN to prevacuolar compartment,
but in Sac. cerevisiae both Syn8p and Tlg1p are thought to
function as counterparts of Fsv1p (Takegawa et al., 2003a).
Moreover, Sch. pombe Pep12p functions from the TGN to
the vacuole via the prevacuolar compartment, whereas Sac.
cerevisiae Pep12p plays a role from the TGN to the
prevacuolar compartment and Vam3p covers the function
from the prevacuolar compartment to the vacuole
(Hosomi et al., 2011). This functional relationship can
also be seen in the filamentous fungus Aspergillus oryzae,
where AoVam3p is the sole orthologue of both Sac.
cerevisiae Vam3p and Pep12p (Shoji et al., 2006; Kuratsu et
al., 2007). Furthermore, although Vsl1p is thought to be
the counterpart of Vam7p as we elucidated here, Vsl1p has
functional redundancy with Fsv1p.
In this study, we added Vsl1p as a member of the SNARE
proteins, and so far we have identified 18 SNARE-related
genes in Sch. pombe. Nevertheless, this total number is less
than the 24 genes reported in Sac. cerevisiae (Pelham,
1999). Since we discovered Vsl1p in this research, more
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Microbiology 161
Sch. pombe Vsl1p in vacuolar protein transport and fusion
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(a)
Sp Vsl1p
341 aa
16 %
Sp Fsv1p
247 aa
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Prevacuole
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Fig. 8. Schematic diagram of the functions of Sch. pombe (Sp)
Vsl1p, Fsv1p and Pep12p. (a) Comparison of the primary
structures of Vsl1p and Fsv1p. The identity between these
proteins is shown. The black, hatched and grey boxes delineate
the PX domain, SNARE motif and transmembrane domain,
respectively. (b) A working hypothesis that draws the functions
of SNARE proteins localizing from Golgi to vacuole through the
prevacuolar compartment. Vsl1p is a partner of Pep12p, and
mainly functions on the prevacuolar and vacuolar membrane.
Fsv1p is also a partner of Pep12p, but primarily works on the Golgi
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Darsow, T., Rieder, S. E. & Emr, S. D. (1997). A multispecificity
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cryptic SNARE proteins might be found in the Sch. pombe
genome. It is a general theory that membrane fusion is
controlled by a combination of three or four SNARE
proteins; therefore, deficiency of even one SNARE protein
can cause serious cellular problems. However, abnormal
vacuolar morphology was observed not in the single deletion mutant vsl1D or fsv1D cells, but in the double deletion mutant fsv1Dvsl1D cells only. These data suggest the
possibility that multiple different SNARE proteins have
redundant functions in Sch. pombe. Further detailed
analyses of Sch. pombe SNARE proteins will provide a
better understanding of the vesicular membrane-trafficking
pathway.
pombe Pep12p is required for vacuolar protein transport and vacuolar
homotypic fusion. J Biosci Bioeng 112, 309–314.
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Iwaki, T., Hosomi, A., Tokudomi, S., Kusunoki, Y., Fujita, Y., GigaHama, Y., Tanaka, N. & Takegawa, K. (2006). Vacuolar protein
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ACKNOWLEDGEMENTS
This study was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture of
Japan (to K. T.).
Kuratsu, M., Taura, A., Shoji, J. Y., Kikuchi, S., Arioka, M. & Kitamoto,
K. (2007). Systematic analysis of SNARE localization in the filamentous
fungus Aspergillus oryzae. Fungal Genet Biol 44, 1310–1323.
Kweon, Y., Rothe, A., Conibear, E. & Stevens, T. H. (2003). Ykt6p is a
multifunctional yeast R-SNARE that is required for multiple
membrane transport pathways to the vacuole. Mol Biol Cell 14,
1868–1881.
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