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Yeast 2003; 20: 845–855.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1011
Research Article
Characterization of vps33+, a gene required for
vacuolar biogenesis and protein sorting in
Schizosaccharomyces pombe
Tomoko Iwaki1 , Fumi Osawa1 , Masayuki Onishi2 , Takako Koga2 , Yasuko Fujita1 , Akira Hosomi1 ,
Naotaka Tanaka1 , Yasuhisa Fukui2 and Kaoru Takegawa1 *
1 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
2 Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan
*Correspondence to:
Kaoru Takegawa, Department of
Life Sciences, Faculty of
Agriculture, Kagawa University,
Miki-cho, Kagawa
761-0795, Japan.
E-mail:
[email protected]
Received: 13 March 2003
Accepted: 12 April 2003
Abstract
From the fission yeast Schizosaccharomyces pombe we have identified and deleted
vps33, a gene encoding a homologue of VPS33, which is required for vacuolar
biogenesis in S. cerevisiae cells. When the vps33+ gene is disrupted, Sz. pombe strains
are temperature-sensitive for growth and contain numerous small vesicular structures
stained with FM4–64 in the cells. Deletion of the Sz. pombe vps33+ gene results
in pleiotropic phenotypes consistent with the absence of normal vacuoles, including
missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and
sporulation defects. These results are consistent with Vps33p being necessary for the
morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz.
pombe cells. Copyright  2003 John Wiley & Sons, Ltd.
Keywords: vacuolar protein sorting; Schizosaccharomyces pombe; vacuolar biogenesis; sporulation
Introduction
Eukaryotic cells are characterized by a number
of different membrane-bound compartments, which
serve special roles in the cell. In the yeast Saccharomyces cerevisiae, the vacuole is the most prominent organelle with significant morphology: it is
distinguished by its large volume of about onequarter of the total cell volume. The vacuole of
S. cerevisiae is an acidic organelle and its internal
pH is maintained at 6.2 by a vacuolar-type proteinpumping ATPase. The vacuole is also involved in a
variety of physiological processes, such as protein
turnover, ion and pH homeostasis and osmoregulation (Jones et al., 1997).
To identify the transport components involved
in the delivery of proteins to the vacuole, genetic
selections in S. cerevisiae were used to isolate
mutants that missort and secrete the vacuolar
hydrolase carboxypeptidase Y (CPY) (Bankaitis
Copyright  2003 John Wiley & Sons, Ltd.
et al., 1986; Rothman and Stevens, 1986). Many of
the resulting vacuolar protein sorting (vps) mutants
were also isolated with screens for vacuolar peptidase deficiencies (pep; Jones, 1977) and vacuolar morphological defects (vam; Wada et al.,
1992). Together, these mutants define more than
40 complementation groups and have been categorized into six classes (A–F) with respect to
their vacuolar protein sorting, morphology and
acidification defects (Banta et al., 1988; Raymond et al., 1992). The four class C vps mutants
(pep5/vps11/end1/vam1, vps16/vam9, pep3/vps18/
vam8, and vps33/slp1/vam5/pep14/met27 ) exhibit
the most severe vacuolar protein sorting and morphological defects. The class C vps mutants lack
any structure resembling a normal vacuole and
instead accumulate small vesicles and other aberrant membrane compartments (Banta et al., 1988;
Woolford et al., 1990; Preston et al., 1991). Their
gene products physically associate together to form
846
the class C Vps protein complex on the vacuolar
membrane (Rieder and Emr, 1997). The class C
Vps protein complex plays essential roles in the
processes of membrane docking and fusion at both
the Golgi-to-endosome and endosome-to-vacuole
stages of transport (Wurmser et al., 2000; Srivastava et al., 2000; Sato et al., 2000; Peterson and
Emr, 2001).
The fission yeast Schizosaccharomyces pombe,
taxonomically and evolutionally distant from the
budding yeast (Russell and Nurse, 1986), is genetically and physiologically well characterized (Egel
et al., 1980). Under normal conditions, Sz. pombe
has a large number of small vacuoles, unlike S.
cerevisiae. Although the functional roles of class C
Vps proteins have been extensively investigated in
S. cerevisiae, little is known about their Sz. pombe
counterparts. Moreover, there are no reports on the
isolation and characterization of vacuole-deficient
mutants in Sz. pombe. Therefore, we sought to
identify and characterize class C Vps proteins that
may control vacuolar biogenesis and protein sorting events in Sz. pombe cells. One of the Class C
VPS genes, VPS33/SLP1, encodes a Sec1p homologue of 691 amino acids (Banta et al., 1990; Wada
et al., 1990). The genome sequence of fission yeast
has recently been reported (Wood et al., 2002). We
found by a BLAST search of protein databases that
Sz. pombe contains a gene in its chromosome II
(SPBC1703.15c) that is homologous to Vps33p of
S. cerevisiae.
In an effort to understand the role of vacuoles
in fission yeast cells better, we have constructed
a strain with a disrupted vps33 gene and characterized its physiological and morphological phenotypes. This is the first report on the isolation
and characterization of a vacuolar-deficient mutant
from the fission yeast. Based on our findings,
we suggest that the Sz. pombe Vps33 protein is
required for multiple vacuolar functions, including
vacuolar fusion and biogenesis events.
Materials and methods
Strains, media, and genetic methods
Escherichia coli XL1-blue (Stratagene) was used
for all cloning procedures. The wild-type Sz. pombe
strains TP4–5A (h- leu1 ura4-D18 ade6-M210 )
and TP4–1D (h + leu1 ura4-D18 his2 ura4 ade6M216 ) (Ohkura et al., 1989) were obtained from
Copyright  2003 John Wiley & Sons, Ltd.
T. Iwaki et al.
Dr T. Toda (ICRF, UK), and KJ100–7B (h 90 leu1
ura4 ) was obtained from Dr K. Tanaka (Tokyo
University, Japan). The cpy1 (h + leu1–32 his2
ura4-D18 ade6-M216 cpy1::ura4+ ) and vps34
(h + leu1–32 ura4-D18 ade6-M216 vps34::ura4+ )
mutants were constructed as described previously
(Tabuchi et al., 1997; Takegawa et al., 1995). Standard rich medium containing 5 g/l yeast extract,
30 g/l glucose and 250 mg/l adenine sulphate
(YEA), 10 g/l yeast extract, 5 g/l peptone and
20 g/l glucose (YPD), synthetic minimal medium
(MM) and sporulation medium (ME) for Sz. pombe
cells were used, as described elsewhere (Moreno
et al., 1991). Sz. pombe cells were transformed by
the lithium acetate method or electroporation, as
described previously (Okazaki et al., 1990; Suga
et al., 2000; Suga and Hatakeyama, 2001). The
general genetic methods have been described previously (Alfa et al., 1993).
Plasmid constructions
pREP41-Hmt1–GFP was constructed as follows.
The hmt1 cDNA clone was obtained from Dr D.W.
Ow (UC Berkeley, USA). XhoI and NotI sites
were introduced at the 5 and 3 ends, and two
oligonucleotides were used to amplify hmt1 by
PCR. The corresponding PCR product was digested
with XhoI and NotI and cloned into the corresponding site of pTN197 derived from pREP41 (Nakamura et al., 2001). The cells were observed with
an Olympus BX-60 fluorescence microscope using
a U-MGFPHQ filter set (Olympus, Tokyo, Japan).
Images were captured with a Sensys Cooled CCD
camera using a MetaMorph (Roper Scientific, San
Diego, CA), and were saved as Adobe Photoshop
files on a Macintosh G4 computer.
Gene disruptions
The vps33+ locus was disrupted in the wildtype Sz. pombe strain by replacing an internal
vps33 gene fragment with the Sz. pombe ura4
gene. To amplify the DNA fragment of vps33
from chromosomal DNA of Sz. pombe, the following oligonucleotides were synthesized: sense,
5 -GCAGACGTCCTAAAGAGTTGGTGG-3 and
antisense, 5 -AATCTCTCCTTCCACCTTCCC-3 .
A fragment of 0.7 kbp was recovered, and ligated into pGEM-T EASY vector (Promega). A
Yeast 2003; 20: 845–855.
Characterization of Sz. pombe vacuole-deficient mutants
HindIII site within the cloned vps33+ open reading frame was digested and a 1.6 kb ura4+ cassette
(Grimm et al., 1988) was inserted. A linearized
DNA fragment carrying this disrupted vps33+ gene
was used to transform a wild-type haploid TP4–1D
(h + ) and KJ100–7B (h 90 ), and ura+ transformants were selected. To confirm that one of the
vps33+ genes had been disrupted, ura+ transformants were analysed by Southern blotting and
PCR to verify correct integration of the deletion
constructs.
The Sz. pombe ypt7+ gene (SPBC405.04c) was
amplified from chromosomal DNA, and the Bgl II
site within the ypt7+ open reading frame was
digested and the ura4+ gene was inserted. A
linearized DNA fragment carrying this disrupted
ypt7+ gene was used to transform a wild-type
haploid TP4–1D, and the disruption was analysed
by PCR.
Vacuole staining
Vacuoles were labelled with FM4–64 (Vida and
Emr, 1995). Cells were grown to exponential
phase in YES medium at 27 ◦ C. Samples of
250 µl cells were incubated with medium containing 80 µM FM4–64 for 30 min at 27 ◦ C. The
cells were then centrifuged at 13 000 × g for
1 min, washed by resuspending them in YES to
remove free FM4–64, and collected by centrifugation at 13 000 × g for 1 min. Cells were then
resuspended in YES and incubated for 90 min at
27 ◦ C before microscopic observation. Stained cells
were observed under a fluorescence microscope
(Model BX-60; Olympus) and Sensys Cooled CCD
camera.
Pulse-chase analysis and immunoblot analysis of
the Sz. pombe Cpy protein
Pulse-chase analysis and immunoprecipitation of
the vacuolar carboxypeptidase Y from Sz. pombe
(SpCPY) were carried out as previously described
(Tabuchi et al., 1997). Antibody incubations were
carried out using rabbit polyclonal antibody against
Sz. pombe Cpy1p (Tabuchi et al., 1997). The
SpCPY colony blot assay was performed by
replica-plating freshly grown spots onto nitrocellulose for 3 days of growth, as previously described
(Cheng et al., 2002).
Copyright  2003 John Wiley & Sons, Ltd.
847
Results
Isolation and disruption of the fission yeast
VPS33 homologue gene
An examination of the Sz. pombe genome
database revealed that one gene in chromosome II
(SPBC1703.15c) is predicted to encode a protein
homologous to the S. cerevisiae Vps33 protein.
Therefore we designated this gene vps33. The
vps33+ gene encodes a hydrophilic protein of
593 amino acids with three introns. A BLAST
search comparing the sequences of Sz. pombe
and S. cerevisiae Vps33 proteins to the GenBank
database revealed that the protein is highly
homologous to proteins found in higher eukaryotes,
e.g. human (Carim et al., 2000; Huizing et al.,
2001), rat (Pevsner et al., 1996), fly, nematode
and plant (Huizing et al., 2001). Figure 1A shows
the result of a sequence alignment through the
region of highest homology for Vps33p. Notably,
the degree of homology was highest at residues
214–243 of Sz. pombe Vps33p, and this region
is also highly homologous to another Sec1 family
protein, Vps45p from budding yeast (Piper et al.,
1994; Cowles et al., 1994), plant (Bassham and
Raikhel, 1998) and human (Pevsner et al., 1996)
(Figure 1A). The conservation of the Vps33 protein
between organisms as widely divergent as yeast
and humans indicates that this protein may play
a fundamental role in maintaining proper protein
trafficking within eukaryotic cells.
To examine the phenotypic consequences of
a null allele of vps33+ , we performed a gene
disruption of this locus. A linear fragment of the
vps33+ gene inserted with the Sz. pombe ura4
gene was used to transform haploid strain TP4–1D
(Figure 1B). Several transformants (slow-growing
colonies) were isolated and the structure of the
disrupted allele was verified by Southern blot and
PCR analyses (data not shown).
Sz. pombe vps33 disruption affects cell growth
and vacuole morphology
Although the Sz. pombe vps33+ is not essential
at low temperatures, the growth of the vps33disrupted cells was very slow. Therefore the growth
properties of vps33 cells were analysed further.
In liquid YES medium at 27 ◦ C, the cells had a
doubling time of ∼8 h, in contrast to 2 h 30 min for
wild-type cells. The disruptants showed only slight
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T. Iwaki et al.
A
28%
196 276 305
29% Identity
526
A
642 689
691aa
ScVps33p
138 214
243
465
539
vps33 ∆
582
592aa
SpVps33p
(51%)
214
WT
243
26°C
B
Nomarski
37°C
FM4-64
WT
B
P
H
EH
EHHH
vps33 ∆
ura4+
0.5 kb
Figure 1. (A) The Vps33 protein is highly conserved in
eukaryotes. The most highly conserved residues 214–243
of Sz. pombe Vps33p are aligned to show the residues of
greatest homology between the budding yeast (ScVps33p),
rat (RnVps33p), plant (AtVps33p) and human (HsVps33p)
homologues. These residues are also aligned between the
budding yeast (ScVps45p), plant (AtVps45p) and human
(HsVps45p) Sec1 family Vps45 protein. (B) Disruption of
vps33+ . A restriction map of the vps33+ gene is shown
with the open reading frame indicated by an open arrow.
Restriction sites: H, HindIII; E, EcoRI; P, PstI
Figure 2. (A) The growth phenotypes of wild-type and
vps33 cells on YES plates at 26 ◦ C and 37 ◦ C. Plates
were incubated at the indicated temperature for 5 days.
(B) Vacuolar morphology in wild-type and vps33 cells.
Wild-type (WT) and vps33 cells were grown in YES at
27 ◦ C to mid-log phase and stained with FM4–64 by the
method described in Materials and methods. The stained
cells were then visualized using Nomarski optics (left panels)
and fluorescence microscopy (right panels)
growth on synthetic minimal medium. We found
that addition of excess NH4 Cl (final concentration
of 94 mM; Alfa et al., 1993) to MM medium
inhibits the growth of vps33 cells, and additions
of 40 mM NH4 Cl were appropriate for the growth
(data not shown). This mechanism is unknown but
for vps33 mutants it appears to correlate with
defects of vacuolar functions.
Colonies of wild-type (TP4–1D) and vps33
cells were streaked onto YES plates and incubated
at 26 ◦ C and 37 ◦ C for 4 days. While both wildtype and vps33 cells grew at 26 ◦ C, vps33
cells exhibited a temperature-sensitive growth at
37 ◦ C (Figure 2A). This result is similar to that
observed in S. cerevisiae vps33 strains (Banta
et al., 1990); disruption of the VPS33 gene renders
cells unable to survive at 37 ◦ C, which may result
from decreased tolerance to temperature stress due
to abnormal vacuolar function.
In S. cerevisiae, cells in the vps33 mutant
exhibited extreme defects in vacuole biogenesis
and accumulated vesicles and membrane-enclosed
compartments that bore no resemblance to a normal vacuole. To determine the effect of the vps33+
null mutation on vacuole structure in Sz. pombe
cells, a vital stain for the vacuolar membrane, FM
4–64, was used in microscopic examination of
wild-type and vps33 cells. The cell-shape and
septum formation of the vps33 cells were found
to be normal using Nomarski optics. Wild-type
Sz. pombe cells contained fragmented vacuoles,
and these vacuoles appeared to be randomly dispersed throughout the cells. In contrast, cells carrying the vps33 null mutation lacked any structures resembling the vacuoles seen in the wild-type
cells under fluorescence optics. The vps33 mutant
cells contained numerous small vesicular structures
stained with FM4–64 (Figure 2B). These small
vesicular structures may be reminiscent of prevacuolar or prelysosomal compartments like S. cerevisiae vps33 mutants (Rieder and Emr, 1997; Vida
and Gerhardt, 2000). Together, these experiments
Copyright  2003 John Wiley & Sons, Ltd.
Yeast 2003; 20: 845–855.
Characterization of Sz. pombe vacuole-deficient mutants
demonstrate that loss of Sz. pombe Vps33p function in the vps33 strain had a significant impact
on the growth and vacuolar morphology of these
cells.
849
Nomarski
FM4-64
WT
The Sz. pombe Vps33 protein is required for
vacuolar fusion in vivo
Isolated vacuoles from S. cerevisiae can undergo
fusion in vitro (Wickner, 2002; Wickner and Haas,
2000). One factor required for this process is the
class C Vps complex (Sato et al., 2000). Under
normal conditions, Sz. pombe has a large number
of small vacuoles, and hypotonic stress caused
transitory fusion of vacuoles (Bone et al., 1998).
Wild-type and vps33 cells, as well as ypt7 cells,
which are deficient in vacuolar fusion (Bone et al.,
1998), were grown in YES media, stained with
FM4–64, and shifted into water to observe the
vacuolar morphology (Figure 3). Wild-type cells
had a smaller number of larger vacuoles that
resulted from vacuolar fusion. Vacuoles of ypt7
cells are smaller than those of wild-type cells
grown in YES medium and vacuolar fusion was
not induced by osmotic stress, indicating that Ypt7p
is required for the vacuolar fusion process like in
S. cerevisiae. The vacuolar structures of vps33
cells were not detected after the shift into water,
suggesting that the vacuolar fusion in vivo induced
by hypotonic stress is dependent on Vps33p.
The vps33 mutants show pleiotropic
phenotypes
Disruption of S. cerevisiae VPS33 genes causes
pleiotropic phenotypes, i.e. hypersensitivity to various divalent cations including calcium ion (CLS14
is allelic to VPS33 ; Wada et al., 1992). We examined the sensitivity to divalent cations, finding that
the Sz. pombe vps33 cells show strong sensitivity to 100 mM CaCl2 (Figure 4A), 10 mM MnCl2 ,
5 mM ZnCl2 and 300 mM MgCl2 . The vps33 cells
also show sensitivity to various drugs, e.g. nystatin
at 5 µg/ml and 4 mM sodium orthovanadate.
Disruption of the S. cerevisiae VPS33 gene
results in a methionine requirement phenotype
(Jacquemin-Faure et al., 1994). To determine whether Sz. pombe vps33 strains show methionine
auxotropic phenotype, wild-type and vps33 cells
were grown on MM media in the presence and
absence of methionine at 75 µg/ml. The vps33
cells grew well on both MM + Met and MM − Met
Copyright  2003 John Wiley & Sons, Ltd.
ypt7∆
vps33∆
Figure 3. The vps33 cells are defective in vacuole fusion
in vivo. Wild-type (WT), ypt7 and vps33 cells were grown
in YES at 27 ◦ C and stained by FM4–64. Cells were shifted
to water for 60 min and the cells were then visualized using
Nomarski optics and fluorescence microscopy
media at 27 ◦ C for 4 days (Figure 4B). This result
shows that Sz. pombe vps33 mutants do not
require methionine for growth, unlike S. cerevisiae
vps33 mutants.
Defects in the ade6+ gene of Sz. pombe (equivalent to the ADE2 gene of S. cerevisiae) leads to
accumulation of the purine nucleotide intermediate,
phosphoribosylaminoimidazole (Chaudhuri et al.,
1996) and consequently their colonies are red. In
Sz. pombe and S. cerevisiae cells, this red pigment is accumulated in the vacuoles when limiting
amounts of adenine are supplied (Chaudhuri et al.,
1996; Weisman et al., 1987; Pringle et al., 1989).
This pigment is known to be fluorogenic and so the
vacuolar compartments can be seen with a fluorescence microscope. The wild-type TP4–1D (ade6M216 ) cells accumulated red pigment in their vacuoles when grown on YPD medium, especially in
the stationary phase (Figure 4C). In contrast, the
vps33 mutant cells did not show any compartments
stained with ade fluorochrome (Figure 4C).
The fission yeast exists in two mating types,
h + (plus) and h − (minus) (Gutz et al., 1974), and
Yeast 2003; 20: 845–855.
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T. Iwaki et al.
After incubation for 3 days, >90% of the wild-type
(h 90 ) cells showed the formation of zygotes and
sporulation, while the vps33 cells showed no formation of zygotes (Figure 5). These results suggest
that the vps33 gene is required for efficient mating.
A
WT
vps33∆
CaCl2
Localization of vacuolar proteins in the vps33
mutants
B
WT
vps33∆
-Met
Nomarski
C
+Met
We have previously reported the isolation and
characterization of a vacuolar marker protein,
carboxypeptidase Y from Sz. pombe (SpCpy1p)
(Tabuchi et al., 1997). We examined the sorting
of SpCpy1p in the vps33 strain. During
Fluorescence
WT
WT
vps33∆
Figure 4. (A) The vps33 cells are sensitive to calcium
ion. Wild-type (WT) and vps33 cells were incubated on
YES plates containing 100 mM CaCl2 for 4 days at 28 ◦ C.
(B) Growth of wild-type and vps33 cells on minimal plates
in the absence (MM — Met) or presence (MM + Met) of
75 µg/ml methionine. (C) Adenine intermediates did not
accumulate in vps33 cells. Wild-type and vps33 cells
were grown to stationary phase in YPD at 27 ◦ C, and then
visualized using fluorescence microscopy
vps33∆
when haploid cells of opposite types are shifted
to nitrogen-free medium they conjugate to form
a diploid zygote, which then undergoes meiosis
and sporulation (Egel, 1989). Many sporulationdeficient mutants have been isolated and characterized in Sz. pombe (Bresch et al., 1968; Kishida
and Shimoda, 1986) and most of these have exhibited defects in the regulation of the distinct steps of
sporulation or meiosis. To confirm the influence of
the single vps33+ disruption, it was introduced into
a homothallic (h 90 ) strain, which was then plated
onto MEA media and observed microscopically.
Copyright  2003 John Wiley & Sons, Ltd.
Figure 5. Mating deficiency of the homothallic strain with
a disrupted vps33 gene. Wild-type (WT) KJ100–7B (h90
leu1 ura4) and vps33 (h90 leu1 ura4 vps33::ura4+ ) cells
were cultured on ME plates at 28 ◦ C for 3 days, and then
observed using Nomarski optics
Yeast 2003; 20: 845–855.
Characterization of Sz. pombe vacuole-deficient mutants
the synthesis, SpCpy1p undergoes characteristic
modifications and changes in its apparent molecular
mass; after the 15 min pulse period, the ERand Golgi-specific precursor form (proCPY) and
a small amount of the vacuole-specific mature
form (mCPY) were labelled in the wild-type
cells, and after the 30 min chase, all SpCPY
had been transported to the vacuole and matured
(Figure 6A). The vps33 null mutant showed a
severe sorting defect for SpCPY. After the 30 min
chase, mCPY was not detected in the vps33 cells
(Figure 6A). To confirm the missort of SpCPY to
the cell surface in the vps33 cells, we employed
the CPY colony blot assay that directly tests cells
for secretion of SpCPY. In wild-type cells, SpCPY
is efficiently sorted to the vacuole and therefore
is not secreted. In contrast, the vps33 cells
showed strong secretion of SpCPY (Figure 6B).
These results indicate that the Vps33 protein is
required for SpCPY delivery to the vacuole in Sz.
pombe.
The Sz. pombe cells detoxify excessive cadmium by synthesizing phytochelatins, which bind
cadmium and mediate its sequestration into
the vacuole (Cobbett, 2000). Cadmium-sensitive
mutants deficient in the accumulation of a phytochelatin–cadmium complex were isolated from
Sz. pombe (Ortiz et al., 1992). One of the genes,
hmt1+ , encodes an ABC-type transporter protein required for cadmium tolerance (Ortiz et al.,
1992). The Hmt1-β-galactosidase fusion protein
was localized to the Sz. pombe vacuole (Ortiz
et al., 1992), and the phytochelatin–cadmium complex is accumulated in the vacuole by the Hmt1
transporter (Ortiz et al., 1995). We examined the
cadmium sensitivity of the vps33 strain, and
found that the mutant shows strong sensitivity
(Figure 7A). This result suggests that the Hmt1
cadmium transporter may not properly localize
to the vacuolar membrane. We have constructed
the fusion protein GFP–Hmt1 and observed its
localization in the vps33 cells. In wild-type
cells, the fluorescent pattern of GFP–Hmt1 was
found to overlap with the staining of the vacuolar membrane by FM4–64 (Figure 7B), indicating that GFP-Hmt1 resides predominantly on the
vacuolar membrane. The staining pattern of the
fusion protein was diffuse throughout the vps33
cells, but overlapped with FM4–64 localization
(Figure 7B). These results suggest that the vacuolar membrane protein Hmt1p is transported to
Copyright  2003 John Wiley & Sons, Ltd.
851
vps33∆
WT
A
0
30
0
30
Chase (min)
proCPY
mCPY
B
W
T
∆
y1
cp
4∆
s3
vp
3∆
s3
vp
Figure 6. (A) Processing of carboxypeptidase Y in vivo.
Wild-type (WT) and vps33 cells were pulse-labelled with
Express-35 S-label for 10 min at 28 ◦ C and chased for 30 min.
The immunoprecipitates were separated on an SDS-10%
polyacrylamide gel. The autoradiograms of the fixed dried
gels are shown. The positions of proCPY (110 kDa) and
mature Cpy (mCPY; 32 kDa) are indicated. (B) Immunoblot
analysis of carboxypeptidase Y. Cells were grown on
a nitrocellulose filter overnight at 30 ◦ C and the filter
was processed for immunoblotting using rabbit polyclonal
antibody against Sz. pombe Cpy1p. cpy1 was used as a
negative control and vps34 was used as a positive control
for Cpy1p missorting
the prevacuolar compartment; however, the vacuolar pool for the phytochelatin–cadmium complex is significantly reduced because the volume of
the vacuolar compartment is reduced by the vps33
deletion.
Discussion
In an effort to determine proteins involved in
vacuolar biogenesis, we identified the Sz. pombe
vps33+ gene homologous to S. cerevisiae VPS33.
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T. Iwaki et al.
A
WT
vps33∆
0.1 mM CdCl2
WT
B
vps33∆
Nomarski
Hmt1-GFP
FM4-64
Merge
Figure 7. (A) The vps33 cells are sensitive to cadmium
ions. Wild-type (WT) and vps33 cells were incubated on
YES plates containing 0.1 mM CdCl2 for 4 days at 28 ◦ C.
(B) Intracellular localization of Hmt1–GFP. Wild-type and
vps33 cells were grown in MM supplemented with 5 µg/ml
thiamine medium at 27 ◦ C. The cells were harvested and
grown in MM — thiamine medium for 12 h, and stained by
FM4–64. The cells were incubated with water for 2 h to
induce vacuolar fusion. The Hmt1–GFP fusion protein and
vacuolar morphology stained by FM4–64 were visualized
using Nomarski optics and fluorescence microscopy
According to the Sz. pombe GeneDB operated
by the Sanger Institute (http://www.genedb.org/
Copyright  2003 John Wiley & Sons, Ltd.
genedb/pombe/index.jsp), the Sz. pombe vps33+
gene is constitutively expressed throughout the
cell cycle. The constitutive expression of Vps33
suggests that this protein may play a fundamental role in maintaining protein trafficking within
Sz. pombe cells. Disruption of the Sz. pombe
vps33+ gene results in severe and pleiotropic phenotypes consistent with the absence of normal
vacuoles, including temperature-sensitive growth
defects, various ion- sensitivities and sporulation
defects. Although the vacuolar-deficient vps33
cells are viable at low temperatures, the severity
of the vps33 phenotype demonstrates that the presence of a normal vacuole and correct targeting of
cellular material to the vacuole is essential for the
fission yeast cells.
The vacuole participates in the maintenance
of cytoplasmic homeostasis by transporting ionic
molecules across the vacuolar membrane. Therefore, the large volume of the vacuole probably
endows it with a high capacity for the chemiosmotic work that directs ionic homeostasis in the
cytoplasm. Our results indicate that the vacuolardeficient vps33 mutant shows strong sensitivity to various cations that are subject to vacuolar sequestration in S. cerevisiae cells (Bode
et al., 1995). Although the soluble vacuolar protease SpCPY was missorted to the cell surface,
the vacuolar membrane ABC transporter Hmt1 protein was directed to the prevacuolar compartment
in the vps33 cells (Figures 6 and 7). This result
suggests that the vacuolar membrane transporters
may be correctly sorting proteins to the vacuolar membrane in the vps33 cells. Efforts are
under way to identify the sorting mechanism of
putative vacuolar membrane transporters, including V-ATPase and metal transporters in Sz. pombe
cells.
Conjugation of the haploid cells of opposite mating types did not occur in the Sz. pombe vps33
disruptant (Figure 4). Genetic analyses indicate that
vacuolar protease activities play an important role
in sporulation, and vacuolar proteases are required
for the nitrogen starvation-induced protein degradation that accompanies sporulation (Jones, 1977,
1984). Our data show that the vacuolar protease
SpCPY was missorted and secreted to the cell surface in Sz. pombe vps33 cells (Figure 5). We have
also confirmed that a nitrogen starvation-specific
protein Isp6p (Sato et al., 1994), a homologue of
Yeast 2003; 20: 845–855.
Characterization of Sz. pombe vacuole-deficient mutants
vacuolar proteinase B from S. cerevisiae, was significantly secreted to the cell surface in vps33
cells (our unpublished results). Disruption of isp6
arrested the cell cycle prior to conjugation and
caused a drastic blocking effect on spore formation
(Sato et al., 1994). Therefore, the secretion of vacuolar proteases suggests that disruption of vps33
may have marked blocking effects on mating and
spore formation.
The Sz. pombe vps33 cells do not require
methionine for growth, unlike S. cerevisiae vps33
mutants (Figure 3B). The S. cerevisiae met27
mutant was originally isolated as impairing the
transcriptional regulation of the MET genes. Disruption of the MET27 gene leads to a methionine requirement and affects S-adenosyl methionine (AdoMet)-mediated transcriptional control of
genes involved in sulphur metabolism. The cloning
of MET27 showed that it is identical to VPS33
(Jacquemin-Faure et al., 1994). In S. cerevisiae,
the biosynthesis of methionine is controlled by
AdoMet. AdoMet has been shown to be distributed in exchangeable cytosolic and vacuolar
pools, with the latter accounting for up to 70% of
the total vacuolar pools in S. cerevisiae (Schwencke
and de Robichon-Szulmajster, 1976). The lost
of AdoMet storage capacity and increase in the
cytosolic AdoMet concentration modify MET gene
expression, such that addition of methionine to
the growth medium would be required to overcome the imbalance of sulphate flux in S. cerevisiae vps33 cells (Jacquemin-Faure et al., 1994).
Unlike the S. cerevisiae vps33 cells, the Sz. pombe
vps33 cells do not require methionine for growth
(Figure 3B), suggesting that the system of transcriptional regulation may be different between
these two yeasts.
The budding yeast class C Vps complex genetically and biochemically interacts with other proteins: (a) the class B Vps39p (Vam6p) and
Vps41p (Vam2p); (b) Ypt7p, an orthologue to
the mammalian Rab7 GTPase; and (c) a syntaxin
homologue Vam3p (Wickner, 2002). Vps39p is
a guanine–nucleotide exchange factor for Ypt7p
(Wurmser et al., 2000), and the class C Vps complex is also an effector of Ypt7p (Seals et al., 2000).
The Sz. pombe Ypt7 homologue (SpYpt7p) was
identified by PCR on the basis of sequence conservation between S. cerevisiae Ypt7p and mammalian Rab7p (Bone et al., 1998). When Sz. pombe
ypt7 and vps33 cells were transferred from
Copyright  2003 John Wiley & Sons, Ltd.
853
YES medium to water, vacuolar fusion was not
induced by osmotic stress (Figure 3). These results
indicate that both Ypt7p and Vps33p are required
for the vacuolar fusion process, as in S. cerevisiae. Although both proteins are required for the
vacuolar–membrane fusion, the vacuolar morphology of ypt7 cells is relatively normal (Figure 3).
This result suggests that the Vps33 protein plays
additional roles at other intracellular transport
steps.
Members of the Sec1p family of proteins are
thought to regulate membrane fusion through their
direct interaction with syntaxins. The S. cerevisiae
Vps33p genetically and physically associates with
the vacuolar syntaxin homologue Vam3p, and the
class C Vps complex may function in maintaining Vam3p in an unpaired and active state by
preventing non-productive associations on the vacuole (Sato et al., 2000). It is worth pointing out
that no homologue of Vam3p has been found
in Sz. Pombe, although the sequence of the Sz.
pombe genome is complete (Wood et al., 2002).
The identification of Sz. pombe syntaxin homologues involved in vacuolar biogenesis is currently
under way. It is also likely that Sz. pombe Vps33p
also associates with other class C Vps protein-like
molecules. We then inspected the complete genome
database for Sz. pombe in order to search for genes
encoding class C VPS homologues. This inspection
revealed the presence of genes [SPAC823.12 and
SPAC16A10.03c (VPS11 ); SPAC824.05 (VPS16 );
SPCC790.02 (VPS18 )] in Sz. pombe chromosomes.
We are currently examining the role of other Sz.
pombe class C Vps homologues in the events
of vacuolar biogenesis. Characterization of these
mutants will reveal a mechanism for the sorting
of vacuolar protein and vacuolar biogenesis in Sz.
pombe.
Acknowledgements
We would like to thank Drs Takashi Toda, Chikashi
Shimoda, David Ow and Taro Nakamura for providing
the Sz. pombe strains and plasmids. We thank Masaru
Ikeuchi for his excellent technical assistance. This work was
partly supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science and Culture of
Japan and the Project for Development of a Technological
Infrastructure for Industrial Bioprocesses on R&D of New
Industrial Science and Technology Frontiers by the Ministry
of Economy, Trade & Industry (METI), and entrusted
by New Energy and Industrial Technology Development
Organization (NEDO).
Yeast 2003; 20: 845–855.
854
References
Alfa C, Fantes P, Hyams J, McLeod M, Warbrick E. 1993.
Experiments with Fission Yeast: A Laboratory Course Manual.
Cold Spring Harbor Laboratory Press: New York.
Bankaitis VA, Johnson LM, Emr SD. 1986. Isolation of yeast
mutants defective in protein targeting to the vacuole. Proc Natl
Acad Sci USA 83: 9075–9079.
Banta LM, Robinson JS, Klionsky DJ, Emr SD. 1988. Organelle
assembly in yeast: characterization of yeast mutants defective
in vacuolar biogenesis and protein sorting. J Cell Biol 107:
1369–1383.
Banta LM, Vida TA, Herman PK, Emr SD. 1990. Characterization
of yeast Vps33p, a protein required for vacuolar protein sorting
and vacuole biogenesis. Mol Cell Biol 10: 4638–4649.
Bassham DC, Raikhel NV. 1998. An Arabidopsis Vps45p
homologue implicated in protein transport to the vacuole. Plant
Physiol 117: 407–415.
Bode H-P, Dumschat M, Garotti S, Fuhrmann GF. 1995. Iron
sequestration by the yeast vacuole. Eur J Biochem 228:
337–342.
Bone N, Millar JB, Toda T, Armstrong J. 1998. Regulated vacuole
fusion and fission in Schizosaccharomyces pombe: an osmotic
response dependent on MAP kinases. Curr Biol 8: 135–144.
Bresch C, Muller G, Egel R. 1968. Genes involved in meiosis and
sporulation of a yeast. Mol Gen Genet 102: 301–306.
Carim L, Sumoy L, Andreu N, Estivill X, Escarceller M. 2000.
Cloning, mapping and expression analysis of VPS33B, the
human orthologue of rat Vps33b. Cytogenet Cell Genet 89:
92–95.
Chaudhuri B, Ingavale S, Bachhawat AK. 1996. apd1+ , a gene
required for red pigment formation in ade6 mutants of
Schizosaccharomyces pombe, encodes an enzyme required for
glutathione biosynthesis: a role for glutathione and a glutathioneconjugate pump. Genetics 145: 75–83.
Cheng H, Sugiura R, Wu W, et al. 2002. Role of the Rab GTPbinding protein Ypt3 in the fission yeast exocytic pathway,
and its connection to calcineurin function. Mol Biol Cell 13:
2963–2976.
Cobbett CS. 2000. Phytochelatins and their roles in heavy metal
detoxification. Plant Physiol 123: 825–832.
Cowles CR, Emr SD, Horazdovsky BF. 1994. Mutations in the
VPS45 gene, a SEC1 homologue, result in vacuolar protein
sorting defects and accumulation of membrane vesicles. J Cell
Sci 107: 3449–3459.
Egel R. 1989. Mating-type genes, meiosis, and sporulation. In
Molecular Biology of the Fission Yeast, Nasim A, Young P,
Johnson BF (eds). Academic Press: New York; pp 31–73.
Egel R, Kohli J, Thuriaux P, Wolf K. 1980. Genetics of the fission
yeast Schizosaccharomyces pombe. Ann Rev Genet 14: 77–108.
Grimm C, Kohli J, Murray J, Maundrell K. 1988. Genetic
engineering of Schizosaccharomyces pombe: a system for gene
disruption and replacement using the ura4 gene as a selectable
marker. Mol Gen Genet 215: 81–86.
Gutz H, Heslo H, Leupoud L, Loprieno N. 1974. Handbook of
Genetics, vol 1. Plenum: New York; 395–446.
Huizing M, Didier A, Walenta J, et al. 2001. Molecular cloning
and characterization of human VPS18, VPS11, VPS16 and
VPS33. Gene 264: 241–247.
Jacquemin-Faure I, Thomas D, Laporte J, Cibert C, SurdinKerjan Y. 1994. The vacuolar compartment is required for
Copyright  2003 John Wiley & Sons, Ltd.
T. Iwaki et al.
sulphur amino acid homeostasis in Saccharomyces cerevisiae.
Mol Gen Genet 244: 519–529.
Jones EW. 1977. Proteinase mutants of Saccharomyces cerevisiae.
Genetics 85: 23–33.
Jones EW. 1984. The synthesis and function of proteases in
Saccharomyces cerevisiae: genetic approaches. Ann Rev Genet
18: 233–270.
Jones EW, Webb GC, Hiller MA. 1997. Molecular Biology of the
Yeast Saccharomyces, vol III. Cold Spring Harbor Laboratory
Press: New York; 363–469.
Kishida M, Shimoda C. 1986. Genetic mapping of 11 spo
genes essential for ascospore formation in the fission yeast
Schizosaccharomyces pombe. Curr Genet 10: 443–447.
Moreno S, Klar A, Nurse P. 1990. Molecular genetic analysis of
fission yeast Schizosaccharomyces pombe. Methods Enzymol
194: 795–823.
Nakamura T, Nakamura-Kubo M, Hirata A, Shimoda C. 2001.
The Schizosaccharomyces pombe spo3+ gene is required for
assembly of the forespore membrane and genetically interacts
with psy1+ -encoding syntaxin-like protein. Mol Biol Cell 12:
3955–3972.
Ohkura H, Kinoshita N, Miyatani S, Toda T, Yanagida M. 1989.
The fission yeast dis2+ gene required for chromosome disjoining
encodes one of two putative type 1 protein phosphatases. Cell
57: 997–1007.
Okazaki K, Okazaki N, Kume K, et al. 1990. High-frequency
transformation method and library transducing vectors for
cloning mammalian cDNAs by trans-complementation of
Schizosaccharomyces pombe. Nucleic Acids Res 18: 6485–6489.
Ortiz DF, Kreppel L, Speiser DM, et al. 1992. Heavy metal
tolerance in the fission yeast requires an ATP-binding cassettetype vacuolar membrane transporter. EMBO J 11: 3491–3499.
Ortiz DF, Ruscitti T, McCue KF, Ow DW. 1995. Transport of
metal-binding peptides by HMT1, a fission yeast ABC-type
vacuolar membrane protein. J Biol Chem 270: 4721–4728.
Pevsner J, Hsu SC, Hyde PS, Scheller RH. 1996. Mammalian
homologues of yeast vacuolar protein sorting (vps) genes
implicated in Golgi-to-lysosome trafficking. Gene 183: 7–14.
Piper RC, Whitters EA, Stevens TH. 1994. Yeast Vps45p is a
Sec1p-like protein required for the consumption of vacuoletargeted, post-Golgi transport vesicles. Eur J Cell Biol 65:
305–318.
Peterson MR, Emr SD. 2001. The class C Vps complex functions
at multiple stages of the vacuolar transport pathway. Traffic 2:
476–486.
Preston RA, Manolson MF, Becherer K, et al. 1991. Isolation
and characterization of PEP3, a gene required for vacuolar
biogenesis in Saccharomyces cerevisiae. Mol Cell Biol 11:
5801–5812.
Pringle JR, Preston RA, Adams AE, et al. 1989. Fluorescence
microscopy methods for yeast. Methods Cell Biol 31: 357–435.
Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. 1992.
Morphological classification of the yeast vacuolar protein sorting
mutants: evidence for a prevacuolar compartment in class E vps
mutants. Mol Biol Cell 3: 1389–1402.
Rieder SE, Emr SD. 1997. A novel RING finger protein complex
essential for a late step in protein transport to the yeast vacuole.
Mol Biol Cell 8: 2307–2327.
Rothman JH, Stevens TH. 1986. Protein sorting in yeast: mutants
defective in vacuole biogenesis mislocalize vacuolar proteins
into the late secretory pathway. Cell 47: 1041–1051.
Yeast 2003; 20: 845–855.
Characterization of Sz. pombe vacuole-deficient mutants
Russell P, Nurse P. 1986. Schizosaccharomyces pombe and
Saccharomyces cerevisiae: a look at yeasts divided. Cell 45:
781–782.
Sato TK, Rehling P, Peterson MR, Emr SD. 2000. Class C Vps
protein complex regulates vacuolar SNARE pairing and is
required for vesicle docking/fusion. Mol Cell 6: 661–671.
Sato S, Suzuki H, Widyastuti U, Hotta Y, Tabata S. 1994.
Identification and characterization of genes induced during
sexual differentiation in Schizosaccharomyces pombe. Curr
Genet 26: 31–37.
Schwencke J, De Robichon-Szulmajster H. 1976. The transport
of S-adenosyl-L-methionine in isolated yeast vacuoles and
spheroplasts. Eur J Biochem 65: 49–60.
Seals DF, Eitzen G, Margolis N, Wickner WT, Price A. 2000.
A Ypt/Rab effector complex containing the Sec1 homologue
Vps33p is required for homotypic vacuole fusion. Proc Natl
Acad Sci USA 97: 9402–9047.
Srivastava A, Woolford CA, Jones EW. 2000. Pep3p/Pep5p
complex: a putative docking factor at multiple steps of vesicular
transport to the vacuole of Saccharomyces cerevisiae. Genetics
156: 105–122.
Suga M, Hatakeyama T. 2001. High efficiency transformation of
Schizosaccharomyces pombe pretreated with thiol compounds
by electroporation. Yeast 18: 1015–1021.
Suga M, Isobe M, Hatakeyama T. 2000. Cryopreservation of
competent intact yeast cells for efficient electroporation. Yeast
16: 889–896.
Tabuchi M, Iwaihara O, Ohtani Y, et al. 1997. Vacuolar protein
sorting in fission yeast: cloning, biosynthesis, transport and
processing of carboxypeptidase Y from Schizosaccharomyces
pombe. J Bacteriol 179: 4179–4189.
Takegawa K, DeWald DB, Emr SD. 1995. Schizosaccharomyces
pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase
essential for normal cell growth and vacuole morphology. J Cell
Sci 108: 3745–3756.
Copyright  2003 John Wiley & Sons, Ltd.
855
Vida TA, Emr SD. 1995. A new vital stain for visualizing vacuolar
membrane dynamics and endocytosis in yeast. J Cell Biol 128:
779–792.
Vida TA, Gerhardt B. 1999. A cell-free assay allows reconstitution
of Vps33p-dependent transport to the yeast vacuole/lysosome. J
Cell Biol 146: 85–97.
Wada Y, Kitamoto K, Kanbe T, Tanaka K, Anraku Y. 1990. The
SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar
morphogenesis and function. Mol Cell Biol 10: 2214–2223.
Wada Y, Ohsumi Y, Anraku Y. 1992. Genes for directing vacuolar
morphogenesis in Saccharomyces cerevisiae I. Isolation and
characterization of two classes of vam mutants. J Biol Chem
267: 18 665–18 670.
Weisman LS, Bacallao R, Wickner W. 1987. Multiple methods
of visualizing the yeast vacuole permit evaluation of its
morphology and inheritance during the cell cycle. J Cell Biol
105: 1539–1547.
Wickner W. 2002. Yeast vacuoles and membrane fusion pathways.
EMBO J 21: 1241–1247.
Wickner W, Haas A. 2000. Yeast homotypic vacuole fusion: a
window on organelle trafficking mechanisms. Ann Rev Biochem
69: 247–275.
Wood V, et al. 2002. The genome sequence of Schizosaccharomyces pombe. Nature 415: 871–880.
Woolford CA, Dixon CK, Manolson MF, Wright R, Jones EW.
1990. Isolation and characterization of PEP5, a gene essential
for vacuolar biogenesis in Saccharomyces cerevisiae. Genetics
125: 739–752.
Wurmser AE, Sato TK, Emr SD. 2000. New component of the
vacuolar class C–Vps complex couples nucleotide exchange on
the Ypt7 GTPase to SNARE-dependent docking and fusion. J
Cell Biol 151: 551–562.
Yeast 2003; 20: 845–855.