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© 2009 John Wiley & Sons A/S
doi: 10.1111/j.1600-0854.2009.00907.x
Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71,
Play Antagonistic Roles in the Regulation of Vacuolar
Morphology
Jun Kashiwazaki1 , Tomoko Iwaki2 , Kaoru
Takegawa2 , Chikashi Shimoda1 and Taro
Nakamura1, *
1 Department
of Biology, Graduate School of Science,
Osaka City University, Sumiyoshi-ku, Osaka 558-8585,
Japan
2 Department of Life Sciences, Faculty of Agriculture,
Kagawa University, Miki-cho, Kagawa 761-0795, Japan
∗Corresponding author: Taro Nakamura,
[email protected]
Small guanine triphosphatases (GTPases) of the Rab
family are key regulators of membrane trafficking
events between the various subcellular compartments in
eukaryotic cells. Rab7 is a conserved protein required in
the late endocytic pathway and in lysosome biogenesis.
A Schizosaccharomyces pombe (S. pombe) homolog
of Rab7, Ypt7, is necessary for trafficking from the
endosome to the vacuole and for homotypic vacuole
fusion. Here, we identified and characterized a second
fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to
the vacuolar membrane. Cells deleted for ypt71+ exhibit
normal growth rates and morphology. Interestingly, a
ypt71 null mutant contains large vacuoles in contrast
with the small fragmented vacuoles found in the ypt7
null mutant. Furthermore, the ypt71 mutation does
not enhance or alleviate the temperature sensitivity
or vacuole fusion defect of ypt7 cells. Like ypt7
cells, overexpression of ypt71+ caused fragmentation
of vacuoles and inhibits vacuole fusion under hypotonic
conditions. Thus, the two S. pombe Rab7 homologs
act antagonistically in regulating vacuolar morphology.
Analysis of a chimeric Ypt7/Ypt71 protein showed that
Rab7-directed vacuole dynamics, fusion versus fission,
largely depends on the medial region of the protein,
including a part of RabSF3/α3-L7.
Key words: fission yeast, GFP, Rab, organelle, vacuole
Received 25 May 2008, revised and accepted for publication 3 March 2009, uncorrected manuscript published
online 4 March 2009
Ras-like guanine nucleotide-binding proteins, termed Ypt
in yeast or Rab in mammals, are highly conserved throughout evolution and contribute to the process of fusing
membrane vesicles with their appropriate target membrane(1–3). Like the SNARE proteins, each Rab protein
has a characteristic distribution on cell membranes and
every organelle has at least one Rab protein on its cytosolic surface. Rab proteins are required for membrane fusion
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by facilitating and regulating the rate of vesicle docking
and association of v-SNAREs and t-SNAREs.
The budding yeast Saccharomyces cerevisiae (S . cerevisiae) has a single Rab7 ortholog, Ypt7 (ScYpt7p),
which has been well characterized. ScYpt7p is localized primarily to the vacuolar membrane, and plays
a dual role in both late endosome-to-vacuole transport and vacuole–vacuole fusion(4–9). ScYpt7p is activated by the guanine-nucleotide exchange factor (GEF),
Vps39p/Vam6p. A guanine triphosphate (GTP)-bound form
of ScYpt7p on the donor membrane associates with the
homotypic fusion and vacuole protein sorting (HOPS)
tethering complex on the acceptor membrane, which is
responsible for vacuole fusion. Following formation of
the trans-SNARE complex, membranes are fused(6,7).
The Schizosaccharomyces pombe (S. pombe) ypt7 + was
obtained by polymerase chain reaction (PCR) with primers
designed on the basis of sequence conservation between
ScYpt7p and its mammalian homolog Rab7(10). Like
ScYpt7p, S. pombe Ypt7 is also required for vacuole fusion
in vivo. Recently, we observed vacuoles in sporulating
cells undergoing extensive homotypic fusion dependent
on Ypt7 function(11). A ypt7 null mutant forms asci less
frequently, and is defective in development of the forespore membrane, which becomes the plasma membrane
in spores(11,12). Taken together, these observations indicate that Ypt7-mediated vacuole fusion is also crucial for
formation and maturation of ascospores in S. pombe.
The vacuole of S. cerevisiae is a relatively large
organelle functionally equivalent to the lysosome of animal
cells(13). Vacuoles regulate cytosolic pH and osmolarity,
degrade macromolecules, and store various intermediary
metabolites such as amino acids. Vacuoles of S. pombe
are small under normal growth conditions, but rapidly fuse
in cells suspended in water in response to hypotonic stress
in order to maintain isotonic concentrations of solutes in
the cytosol(10). Conversely, vacuole fission can be also
induced in response to hypertonic stress such as a high
concentration of NaCl(10). However, little is known about
why the number and size of vacuoles differ among yeast
species or about the mechanism(s) by which vacuolar
morphology is determined.
In the present study, we characterized a second S. pombe
Rab7 homolog, Ypt71. Like Ypt7, Ypt71 is localized to the
vacuolar membrane. Interestingly, a ypt71 null mutant contained large vacuoles in contrast to the small fragmented
vacuoles found in a ypt7 null mutant. Overexpression
of ypt71+ resulted in a number of fragmented vacuoles.
These data suggest that two S. pombe Rab7 homologs
Two Rab7 Homologs in S. pombe
Figure 1: Ypt71 is an S. pombe Rab7 homolog. A) Comparison of amino acid sequences of Ypt71 (UniProt accession number:
Q9HDY0) and S. pombe Ypt7 (O94655) (SpYpt7), S. cerevisiae Ypt7 (P32939) (ScYpt7), Arabidopsis thaliana Rab71 (Q2HIJ2) (AtRab71),
Drosophila melanogaster Rab7 (O76742) (DmRab7) and Homo sapiens Rab7 (P51149) (HsRab7). Dark and light shaded boxes indicate
identical and similar residues in four of six proteins, respectively. Dashes represent gaps in the sequences. G, guanine-base-binding
motif; PM, phosphate/magnesium-binding motif; gray bar, Rab subfamily-specific motifs (RabSF1-4). A prediction of secondary structures
is indicated (α1–4, β1–6 and L1-10). The predicted sites for prenylation are marked by asterisks. Arrows indicate the junctions between
the Ypt7 and Ypt71 chimeras. B) A dendrogram of Rab7 subfamily proteins from various organisms and Ypt family proteins from fission
and budding yeasts calculated by the neighbor joining method(15). The conventional Rab7 group and Ypt71 group are indicated. Sj,
S. japonicus; So, S. octosporus.; Ao, Aspergillus oryzae; Nc, Neurospora crassa. C) A restriction map and construction of null mutant.
The white arrow indicates the direction and region of the ypt71+ ORF. The XbaI-XbaI fragment was used for the disruption.
control vacuolar morphology in an antagonistic manner.
Further, differences in amino acid sequence between
these two GTPases are largely restricted to the highly
conserved Rab7-specific region. To our knowledge, this
is the first report describing an antagonistic relationship
between Rab GTPase paralogs that function in organelle
morphogenesis. Their differential activities might explain
in part, why only fission yeasts have many small vacuoles
compared to other yeasts.
Results
ypt71+ encodes a second Rab7 ortholog in S. pombe
We found a novel Rab7 homolog in the S. pombe genome
sequence database (Sanger Institute, Hinxton, UK), which
shows a high degree of homology to the Ypt7 protein but
which had not been previously characterized. The open
reading frame (ORF) has been named ypt71+ . The ypt71+
gene encodes a protein of 208 amino acid residues with
a deduced molecular mass of 23.4 kDa. Ypt71 shares
59% identity and 78% similarity with S. pombe Ypt7
and 54% identity and 75% similarity with human Rab7
(Figure 1A). Like other Ypt and Rab proteins, Ypt71 has a
Cys-X-Cys sequence at its C-terminus, which is thought
to be modified with geranylgeranyl groups(3,14). We also
found this second Rab7 ortholog in sequence databases
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of other fission yeasts including Schizosaccharomyces
octosporus (S. octosporus) and Schizosaccharomyces
japonicus (S. japonicus) (Broad Institute) (Figure S1).
Phylogenetic analysis(15) shows that the Rab7 family is
divided into two groups; the conventional Rab7 group and
the Ypt71 group (Figure 1B).
To investigate the physiological significance of ypt71+ ,
we carried out a one-step gene disruption of the ypt71+
gene. About 33% of the ORF was deleted and replaced
with the ura4+ gene (Figure 1C). ypt71 null mutants were
viable, and exhibited no significant changes in growth
rate, cell morphology, temperature sensitivity, heat shock
responses, sensitivity to high concentrations of ions such
as Ca2+ and K+ , or osmostress (data not shown). The
ypt7 mutant is known to form few and immature spores
in asci(11,12). Unlike ypt7, ypt71 cells can form spores
normally (data not shown). Furthermore, a ypt71 allele
in a ypt7 background did not enhance or alleviate
the known temperature sensitivity, Ca2+ sensitivity or
sporulation defect (data not shown).
Ypt71 localizes to the vacuolar membrane
We next determined the intracellular distribution of
Ypt71. To this end, we used strain ZK130, in which
green fluorescent protein (GFP)-tagged ypt71+ was
chromosomally integrated and driven by its own promoter.
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Figure 2: Intracellular localization of GFPtagged Ypt71. Cells expressing GFP-Ypt71
(ZK130) were stained with FM4-64, chased in
YE liquid medium for 30 min (YE), and further
incubated in water at room temperature for
60 min (water). Under these conditions, both
the endosome and the vacuolar membrane
were stained. Here, the cells in which the GFPYpt71 and FM4-64 signals appear to overlap on
the vacuolar membrane are shown. Bar, 10 μm.
GFP-Ypt71 was functional because the fusion gene
was able to complement the phenotype of ypt71 as
described below. Microscopic observation revealed a
number of fluorescent ring-like structures surrounding
the nucleus (Figure 2), typical of vacuolar membrane
proteins. To confirm whether GFP-Ypt71 localizes to
the vacuolar membrane, we labeled the membrane
with FM4-64, which is taken up by endocytosis and
eventually transported to the vacuolar membrane via the
endosome(16). The fluorescence of GFP-Ypt71 was found
to overlap with the ring-like structure of the vacuolar
membrane stained with FM4-64 (Figure 2). Whereas, no
GFP-Ypt71 signal was observed in the endosome (data
not shown). These results indicate that, like Ypt7, Ypt71
is localized to the vacuolar membrane. This result also
suggests the possibility that ypt71+ is involved in vacuolar
morphogenesis.
Western analysis revealed that GFP-Ypt71 resolved as
a 48-kDa polypeptide by SDS-PAGE (Figure 3A). This
molecular mass corresponds well with that inferred
from nucleotide sequence data. The amount of Ypt71
was apparently lower than that of Ypt7 (Figure 3A, B).
The amount of both Ypt7 and Ypt71 increased under
hypotonic conditions but relative intensity of Ypt7 to Ypt71
(Ypt7/Ypt71) was nearly constant (Figure 3B). Similar
results were obtained when cells were in stationary
phase (Figure 3B). We next examined the membrane
association of Ypt71. Cell extracts were fractionated
into a membrane fraction and a cytosolic fraction, and
the distribution of Ypt71 was examined in each by
Western blot analysis(17). Most Ypt71 was detected in
the membrane fraction prepared from wild-type cells,
supporting the data obtained by fluorescence microscopy.
About 20% of Ypt71 was detected in the cytosolic fraction
Figure 3: Ypt7 is more abundant than Ypt71.
A and B) Comparison of abundance of Ypt7 and
Ypt71. GFP-tagged ypt7 (ZK134) and GFP-tagged
ypt71 (ZK130) strains were grown in liquid complete
medium (YE 10 or 24 h). Cells at mid-log phase
were transferred to water for 3 h (Water 3 h).
Protein extracts were subjected to western blotting
using an anti-GFP antibody. An anti-Spo14 antibody
was used for the detection of Spo14 (45 kD) as
an internal reference. Quantitation was performed
by using lumino analyzer, LAS1000 and Image
Gauge Software (Fujifilm). The relative intensity
of GFP-Ypt7 or GFP-Ypt71 to Spo14 was indicated
as GFP/Spo14. The relative intensity of GFP-Ypt7
to GFP-Ypt71 was indicated as Ypt7/Ypt71. C)
Subcellular fractionation of Ypt7 and Ypt71. ZK134
and ZK130 strains were grown in YE and then
transferred to water for 3 h. Cells were ruptured,
and subjected to differential centrifugation to
fractionate into a P100 membrane fraction and an
S100 supernatant. Each fraction was resolved by
SDS-PAGE and subjected to western blotting using
an anti-GFP antibody.
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Two Rab7 Homologs in S. pombe
(S100) in both exponentially growing cells and cells under
hypotonic conditions (Figure 3C). In contrast, more than
90% of Ypt7 was detected in the membrane fraction
(P100) in both exponentially growing cells and in cells
under hypotonic conditions (Figure 3C).
Ypt71 and Ypt7 have antagonistic functions
in vacuolar morphogenesis
Either hypotonic conditions or sporulation induces fusion
of vacuoles and Ypt7 is required for this process. Indeed,
ypt7 cells contain fragmented vacuoles(10–12,18). To
determine whether Ypt71 also functions in vacuole
(n = 157)
(n= 130)
fusion, we observed vacuolar morphology in ypt71 cells.
Surprisingly, the size of vacuoles in the ypt71 mutant
was markedly larger than in wild-type cells (Figure 4A).
Vacuolar size was quantified by measuring the diameters of vacuoles. The mean vacuolar diameter in wild-type
and ypt71 cells was 1.16 and 1.58 μm, respectively.
The diameter in ypt7ypt71 cells was indistinguishable
from that observed in ypt7 cells (Figure 4B). Furthermore, the size of the vacuoles in wild-type and ypt71
cells increased under hypotonic conditions. In contrast, the
size was almost constant in ypt7 and ypt7ypt71 cells
(Figure 4C). To assess the effect of overexpression of Ypt7
(n = 132)
(n = 117)
Figure 4: ypt71 null mutants contain remarkably large vacuoles. A) Vacuole morphology of TN4 (WT), ZK104 (ypt71) and ZK102
(ypt7). Cells were incubated on YE medium at 28◦ C for 16 h. Vacuoles were stained with FM4-64. Bar, 10 μm. B) The distribution
of vacuole size in wild-type, ypt71, ypt7 and ypt7ypt71 cells. The diameter of vacuoles was measured by AquaCosmos image
analysis software (Hamamatsu Photonics). The number indicates the mean and standard deviation. C) The size of vacuoles in log phase
culture and cells under hypotonic conditions. The mean and standard deviation are shown.
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Vector
Figure 5: Overexpression of Ypt71 causes
fragmentation of vacuoles. TN4 (WT),
ZK102 (ypt7), ZK104 (ypt71) and ZK106
(ypt7ypt71) transformed with either
pREP1 [vector], pREP1(ypt7) [ypt7 OP ] or
pREP1(ypt71) [ypt71OP ] were incubated on
MM+N at 28◦ C for 16 h. Vacuoles were
stained with FM4-64 in MM+N medium.
Bar, 10 μm. Overexpression of GFP-Ypt7 and
GFP-Ypt71 was confirmed by western analysis using anti-GFP antibody. Overexpression
of GFP-Ypt7 and GFP-Ypt71 caused a similar
effect on vacuole size.
and Ypt71, a wild-type strain was transformed independently with each gene under the control of the thiaminerepressible nmt1+ promoter in the plasmid pREP1.
Interestingly, a number of fragmented vacuoles was
observed in the Ypt71-overexpressing strain (Figure 5).
In contrast, overexpression of Ypt7 caused partial vacuole
enlargement (Figure 5). When placed in water, Ypt71overexpressing cells exhibited no obvious change in vacuolar morphology, suggesting that Ypt71 inhibits vacuole
fusion under hypotonic conditions (Figure 6A).
To test whether the GTP- or GDP-binding ability of Ypt71
is involved in vacuole fragmentation, we constructed
three ypt71 mutants, ypt71-T22N, ypt71-Q67L and ypt71D127A, which are thought to be a dominant negative GDPbound form, a constitutively active GTP-bound form and a
nucleotide-free form, respectively(7,19–21). GTP-binding
blots using various recombinant Ypt71 mutant proteins
showed that, as expected, only wild-type Ypt71 and
Ypt71Q67L were able to bind GTP efficiently (Figure 6B).
These mutants were overexpressed in ypt71 cells.
Surprisingly, fragmented vacuoles were observed in all
mutants (Figure 6A). In other words, all the mutants were
functional. With respect to the vacuole fragmentation
phenotype, the nucleotide-free mutant, Ypt71D127A , was
the most similar to wild-type Ypt71 (Figure 6A). These
data suggest that vacuole fragmentation by Ypt71 does
not require its GTP-binding activity.
Functional differences between Ypt7 and Ypt71
largely depend on the RabSF3/α 3-L7 region
As mentioned above, Ypt71 has a high sequence similarity
with Ypt7, but ypt71 and ypt7 mutants exhibit opposing
phenotypes with respect to vacuolar morphology. To gain
insight into the structural basis for the functional specificity
of these two Rab7 homologs, various chimeric molecules
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were constructed to define the regions of Ypt7 or Ypt71
responsible for their different functions.
First, both proteins were divided into N-terminal and
C-terminal regions. The N-terminal region contains a
guanine base-binding motif, G1; phosphate/magnesiumbinding motifs, PM1-3; and Rab subfamily-specific
regions, RabSF1-3(2,22,23). Switch I (in RabSF2), II,
and α3-L7 (in RabSF3/α3-L7) regions, which are known
to be required for GEF binding(24), are also contained
in the N-terminal region (Figures 1A and 7A). On the
other hand, the C-terminal region includes the other
guanine base-binding motifs, G2 and G3, RabSF4 and
geranylgeranylation site. Swaps were made at conserved
amino acids near each medial point (Figures 1A and
7A). After construction of the chimeras, Ypt7(1–110)71, which contains the N-terminal 110 amino acids of
Ypt7 and the C-terminal 98 amino acids of Ypt71, and
Ypt71(1–110)-7, which contains the N-terminal 110 amino
acids of Ypt71 and the C-terminal 95 amino acids of
Ypt7, by overlapping PCR (see Materials and Methods),
the chimeric genes were cloned into an expression
vector, pREP1, under control of the nmt1 promoter.
These genes were then introduced into ypt7 or ypt71
cells by transformation to assess whether they function
as Ypt7 or Ypt71. Ypt7(1–110)-71 was found able to
partially rescue the vacuole fusion and sporulation defects
of ypt7 (Figure 7B, C, and data not shown) but did
not affect ypt71 phenotypes (Figure 7B). Conversely,
Ypt71(1–110)-7 rescued the ypt71 mutation but not that
of ypt7 (Figure 7B, C). These data indicate that the Nterminal regions of Ypt7 and Ypt71 is important for their
respective functions.
To define the regions of Ypt7 and Ypt71 responsible for
function, a series of chimeric molecules was constructed
(Figure 7A). The chimeric genes then introduced into the
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Two Rab7 Homologs in S. pombe
Vector
Figure 6: Effect of overexpression of various ypt71 mutant genes on vacuolar morphology. A) ZK104 (ypt71) transformed with
either pREP1, pREP1(ypt71), pREP1(ypt71T22N), pREP1(ypt71Q67L) or pREP1(ypt71D127A) were grown in MM liquid medium for 16 h.
Cells were stained with FM4-64 and transferred to water for 60 minutes (Water, 60 min). Bar, 10 μm. Overexpression of GFP-tagged
Ypt71, Ypt71T22N , Ypt71Q67L and Ypt71D127A was confirmed by western analysis using anti-GFP antibody. Overexpression of GFP-Ypt7
and GFP-Ypt71 caused a similar effect on vacuole size. B) Characterization of GST-fused Ypt71 and Ypt71 mutant proteins. The top
image shows a Coomassie brilliant blue (CBB)-stained gel of SDS-PAGE of purified recombinant proteins (see Materials and Methods).
The bottom image shows a filter of the GTP overlay assay. Proteins were transferred onto a nitrocellulose filter, and incubated with
α-[32 P]-GTP (see Materials and Methods).
ypt7 or ypt71 strains and were assessed for function.
Chimeras Ypt71(1–32)-7, Ypt71(1–57)-7 and Ypt71(1–75)7 were able to complement ypt7 phenotypes but
not that those of ypt71. Ypt71(1–90)-7 also almost
completely complemented the ypt7 phenotype and
partially complemented that of ypt71. Ypt7(1–32)-71,
Ypt7(1–57)-71, Ypt7(1–75)-71 and Ypt7(1–90)-71 did not
complement ypt7 While Ypt7(1–32)-71 and Ypt7(1–57)71 almost completely complemented ypt71, Ypt7(1–75)71 and Ypt7(1–90)-71 only partially complemented ypt71
(Figure 7B). These data indicate that the regions from
glutamate-97 to alanine-110 and from methionine-74 to
threonine-110 are important for function of Ypt7 and
Ypt71, respectively (Figure 1A). These regions include
a part of the RabSF3/α3-L7 region, which is known as
one of the Rab subfamily-specific regions required for
interaction with other proteins, including its effector(23).
The RabSF3/α3-L7 region is known to be required for GEF
binding(24).
To map the region more precisely, we constructed an additional series of chimera genes (Figure 8A). Ypt7(32–110)71, Ypt7(57–110)-71 and Ypt7(75–110)-71 complemented
the ypt7 mutation though not as well as wild-type Ypt7.
Ypt7(90–110)-71, which contains only the N-terminal half
of RabSF3/α3-L7 of Ypt7, and Ypt7(RabSF3)-71, which
contains the entire Ypt7 RabSF3/α3-L7 region, partially
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complemented the mutation (Figure 8B, C). These data
suggest that RabSF3/α3-L7 is not sufficient for full function of Ypt7. Furthermore, the β-strand adjacent to the
RabSF3/α3-L7 region (β4) is also important for function.
Similarly, the chimeric genes, Ypt71(32–110)-7,
Ypt71(57–110)-7, Ypt71(75–110)-7 and Ypt71(90–110)-7,
were overexpressed in the ypt71 strain to determine
which among them could provide Ypt71 function
(Figure 9A). While Ypt71(32–110)-7 functioned similarly
to wild-type Ypt71, the other chimeras only partially complemented ypt71. These data indicate that, in addition to
RabSF3/α3-L7, the region from arginine-34 to leucine-56
was also important for Ypt71 function. This region
includes Switch I, which constructs GEF-binding domain
(Figure 1A). Unlike for Ypt7, Ypt71(RabSF3)-7 was unable
to substitute for Ypt71 (Figure 9B, C), suggesting that
the RabSF3/α3-L7 region is essential but not sufficient
for Ypt71 function. Furthermore, Ypt71 requires a wider
region than Ypt7 to function as Ypt71. A comparison
of amino acid sequences for Rab7 homologs among
fission yeasts, S. pombe, S. octosporus and S. japonicus,
allowed us to predict a number of important residues in
Ypt7 or Ypt71 (Figure S1). Some of these amino acids
are located in the Switch I and the RabSF3/α3-L7 regions,
supporting the notion that these regions are important
determinants for Ypt7/Ypt71 function.
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Figure 7: Construction of various chimera genes and their functions (I). A) Schematic illustrations of the constructed chimeras.
White and shaded bars indicate amino acid sequences of Ypt7 and Ypt71, respectively. Black bars indicate guanine nucleotide-binding
motifs, PM1, PM3, G2 and G3. Gray bars indicate effector regions including G1 and PM2 motifs. B) Vacuole size in each mutant
expressing chimeras. ZK102 (ypt7) transformed with the indicated constructs were stained with FM4-64 in YE liquid medium and
transferred to water for 3 h. ZK104 (ypt71) transformed with the indicated constructs were grown in MM liquid medium for 16 h.
Cells were stained with FM4-64 in MM liquid medium. The diameter of the vacuoles was measured by AquaCosmos image analysis
software (Hamamatsu Photonics). The mean and standard deviation are shown. C) ZK102 (ypt7) expressing the indicated constructs
are shown. Bar, 10 μm.
The results of the chimera experiments suggest that, like
other Rab family proteins, Ypt71 function is regulated by
a GEF. In S. cerevisiae, ScYpt7p is activated by the GEF,
ScVps39p/Vam6p, thereby binding to the HOPS tethering
complex on the acceptor membrane. S. pombe has an
ScVps39p ortholog, Vps39. Vacuole fragmentation has
been observed in vps39 cells, suggesting that Vps39
functions as a GEF for Ypt7 (Takegawa unpublished
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data). Vps33 and Vps41 are S. pombe homologs of
S. cerevisiae ScVps33p and ScVps41p, respectively,
components of the HOPS complex(18,25,26). To examine
whether Ypt71 interacts with either Vps33, Vps39 or
Vps41, we performed affinity pull-down experiments
using fission yeast cell lysates and resins decorated with
recombinant glutathione S-transferase (GST)-Rab (Ypt71,
Ypt71T22N , Ypt71Q67L and Ypt71D127A ) fusion proteins.
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Two Rab7 Homologs in S. pombe
Figure 8: Construction of various chimera genes and their functions (II). A) Schematic illustrations of the constructed chimeras.
See Figure 7 legend. B) Vacuole size in ZK102 (ypt7) expressing chimeras. See Figure 7 legend. C) ZK102 (ypt7) expressing the
indicated constructs are shown. Bar, 10 μm.
Figure 9: Construction of various chimera genes and their functions (III). A) Schematic illustrations of the constructed chimeras.
See Figure 7 legend. B) Vacuole size in ZK104 (ypt71) expressing chimeras. See Figure 7 legend. C) ZK104 (ypt71) expressing the
indicated constructs are shown. Bar, 10 μm.
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suggesting that Ypt71 regulates vacuolar morphology
via Ypt7 (Figure 10A). Given that Ypt7 regulates vacuole
fusion(4,6), these data suggest that Ypt71 inhibits vacuole
fusion through inhibition of Ypt7 rather than promoting
vacuole fission (Figure 10A). Although fusion signals
under hypotonic conditions increased the amount of both
Ypt7 and Ypt71, the ratio of Ypt7/Ypt71 was almost
constant (Figure 3B). Therefore, it is unlikely that vacuole
morphology is regulated by the expression level of Ypt7
and Ypt71.
Figure 10: A model for the role of Ypt71 in the regulation of
vacuolar morphology. A) Under normal conditions, the function
of Ypt7 in vacuole fusion is inhibited by Ypt71. Unknown vacuole
fusion signals are presumed to activate Ypt7 function or to inhibit
Ypt71 function resulting in the possibility of vacuole fusion. B)
Speculative molecular mechanism for inhibition of vacuole fusion
by Ypt71.(1) Ypt71 interacts with the HOPS complex to inhibit
vacuole fusion.(2) Ypt71 tightly interacts with GEF to inhibit
activation of Ypt7.
Vps33, Vps39 and Vps41 were retained on either GSTRab fusion proteins but not GST, indicating that Ypt71 can
interact with either protein (Figure S2).
Our chimera experiments revealed that regions conferring
specific functions largely depend on RabSF3/α3-L7 in the
medial region and Switch I and II in the N-terminal region
(Figures 7, 8 and 9). The RabSF3/α3-L7 region is one of the
Rab subfamily-specific regions which can distinguish the
known ten subfamilies of Rab GTPases(22). However, the
functions of the RabSF3/α3-L7 region in the Rab7 family
are not well understood. In mammalian Rab3a, RabSF3/
α3-L7 and RabSF4 are known to be required for interaction
with its effector, Rabphilin-3A(27). Furthermore, Ypt71 can
interact with either Vps33, Vps39 or Vps41. Therefore,
one possibility is that Ypt71 interacts with the HOPS
complex, an effector of Ypt7, thereby inhibiting Ypt7
function (Figure 10B(1)). Alternatively, Ypt71 might inhibit
activation of Ypt7 by tightly interacting with GEF for Ypt7
(Figure 10B(2)) because the RabSF3/α3-L7 and Switch I
regions are also known to be required for binding to
GEF(24). GTP-binding activity was not required for Ypt71
function (Figure 6). On the other hand, Ypt71 is proposed
to bind a GEF in the latter model. In S. cerevisiae,
the nucleotide-free form of ScYpt7 exhibited stronger
interactions with ScVps39, a GEF for ScYpt7, than the
GTP-form and GDP-form ScYpt7(7). Furthermore, all of the
ypt71 mutant proteins were able to interact with either
Vps33, Vps39 or Vps41 (Figure S2). Therefore, model 2
should not be ruled out by the fact that the nucleotide-free
form of Ypt71 is functional. Further analysis is required
to elucidate the molecular mechanism by which vacuolar
morphology is regulated by the two Rab7 proteins.
Discussion
In this study, we identified and characterized a second
S. pombe Rab7 ortholog, ypt71+ . It is known that Ypt7
regulates vacuole fusion and that many smaller vacuoles
are observed in ypt7 cells. Unexpectedly, Ypt7 and Ypt71
were found to play antagonistic roles in the regulation
of vacuolar morphology for the following reasons. First,
vacuoles in ypt71 cells were larger than in wild-type cells.
Second, ypt7 + and ypt71+ were unable to complement
the abnormal vacuolar morphology of ypt71 and ypt7,
respectively (Figure 5). Third, overexpression of Ypt71 in
wild-type cells resulted in formation of smaller vacuoles
as observed in ypt7 cells. Fourth, Ypt71 localized to the
vacuolar membrane like Ypt7.
The function of Ypt71 was completely dependent on Ypt7,
because neither overexpression nor deletion of ypt71+
affected the phenotype of ypt7 cells (Figures 4C and 5),
920
Although it is not clear how vacuoles are fragmented in budding yeast, S. cerevisiae rapidly synthesizes
phosphatydylinositol-3,5-bisphosphate (PI3,5P2 ) under
hyperosmotic conditions by a process that involves activation of a PI3P5 kinase(28). Additionally, the budding yeast
vacuolar casein kinase, Yck3p, is involved in this process.
Phosphorylation of ScVps41p by Yck3p inhibits vacuole
fusion(29). This fact is consistent with our data, in that
vacuole fragmentation is attained by inhibition of vacuole
fusion, rather than enhancement of vacuole fission.
Although ScYpt7p is the sole known Rab7 homolog in
S. cerevisiae, there are many organisms having two or
more Rab7 homologs. In most cases, however, they are
functionally equivalent. In no cases are different orthologs
in a given organism known to function in a co-ordinate
manner. S. cerevisiae Ypt10p is thought to be a Rab5
subfamily member but its role is still unclear (Figure 1B).
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Two Rab7 Homologs in S. pombe
Disruption of YPT10 resulted in no apparent phenotype
while overexpression caused a growth defect(30,31).
Ypt10p and a Rab5 ortholog Vps21p localizes to the
compartments of endocytic pathway(31). The relationship
between Ypt10p and Vps21p resembles that between
Ypt71 and Ypt7, and therefore, Ypt10p may inhibit
functions of Rab5 orthologs. Rab GTPase paralogs that
function in an antagonistic manner may be a general
regulatory mechanism.
Why does S. pombe have a Ypt71-type Rab7 protein in
addition to a ‘conventional’ Rab7, Ypt7? One possibility is
that Ypt71 is required for strict regulation of vacuolar
morphology. An S. pombe cell has numerous small
vacuoles while a budding yeast cell has a few large
vacuoles. Unlike budding yeasts, fission yeasts proliferate
by fission, for which cells must divide in the central
region. The fission yeast ste12 + gene encodes a PI3P5
kinase(32). In ste12 mutants, huge abnormal vacuoles
form, resulting in cells of different sizes in each cell
division(33). For these reasons, the size of the vacuole in
fission yeasts may be kept small. Bone et al.have pointed
out that small vacuoles are important for adaptation
to sudden decreases in osmotic pressure caused for
example, by rainfall(10). As described earlier (Figure 1B),
Ypt71-family proteins are present only in fission yeasts.
Filamentous fungi such as Aspergillus have a few large
vacuoles although its hyphal cells proliferate by fission.
However, the vacuolar morphology of filamentous fungi
is quite different from that of budding and fission yeasts.
Recently, Shoji et al.observed the vacuolar morphology
in greater detail and found that the structure of the
vacuoles changed dramatically from spherical to tubular
shape. They speculated that tubular vacuoles function
as transporters of various substances including proteins
between the compartments of long hyphae(34). Thus,
the mechanisms controlling vacuolar morphology in
filamentous fungi and S. pombe might be different. As
in S. pombe, many small vacuoles were observed in both
S. octosporus and S. japonicus (data not shown). We
speculate that fission yeasts developed a strict and subtle
means of regulating vacuolar morphology by duplicating
a Rab7 gene that subsequently evolved to acquire new
functions.
Materials and Methods
Yeast strains, media and culture conditions
S. pombe strains used in this study are listed in Table 1. Complete
medium YE was used for growth. Synthetic medium MM+N was used
for overexpression. Malt extract medium (MEA) and synthetic sporulation
media (SSA) were used for sporulation(35–37).
Plasmid construction
Plasmid pREP1(ypt71) was constructed as follows. The ypt71+ ORF was
amplified by PCR using ypt71-s and ypt71-as as forward and reverse
primers, respectively (Table S1). Underlined sequences show restriction
enzyme sites. The PCR product was digested with BamHI and Not I,
and then inserted into BamHI- and Not I-digested pREP1(NotI), yielding
Traffic 2009; 10: 912–924
Table 1: Strains used in this study
Name
Genotype
Source
L972
(FY7507)a
TN4
(FY7251)a
TN29
(FY7816)a
KJ100-7BY
(FY12694)a
ZK8
(FY12718)a
ZK43
(FY12752)a
ZK73
(FY12775)a
ZK102
h−
(36)
ZK104
h−
ZK106
h−
ZK116
h90
ZK130
ZK131
h−
h90
ZK134
h−
h (both h−
and h90 )
h90
h90
h−
h90
h−
h−
leu1-32
(38)
ura4-D18 leu1-32
(39)
ypt7::ura4+ ura4-D18
leu1-32
ypt7::ura4+ ura4-D18
(18)
ypt71::ura4+ ura4-D18
leu1-32
ypt71::ura4+ ura4-D18
This study
ypt7::ura4+ ura4-D18
leu1-32
ypt71::ura4+ ura4-D18
leu1-32
ypt7::ura4+
ypt71::ura4+
ura4-D18 leu1-32
ypt71::GFP-ypt71
ura4-D18
ypt71::GFP-ypt71
ypt7::GFP-ypt7
ura4-D18 leu1-32
ypt7::GFP-ypt7
This study
This study
This study
This study
This study
This study
This study
This study
This study
a YGRC,
Yeast Genetic Resource Center Japan (http://yeast.
lab.nig.ac.jp/nig/). The strains constructed in this study will be
deposited in the YGRC.
pREP1(ypt71). Plasmid pREP1(ypt7) was constructed as follows. The
ypt7 + ORF was amplified by PCR using ypt7-s and ypt7-tas as forward and
reverse primers, respectively (Table S1). The PCR product was digested
with XhoI and Sac I, and then inserted into SalI I- and SacI I-digested pREP1,
yielding pREP1(ypt7).
Disruption of ypt71+
ypt71+ was disrupted by replacing a substantial part of the ORF with
ura4+ . A 3-kb XbaI fragment, which contains the ypt71+ ORF, was
amplified by PCR using ypt71-ds and ypt71-das as forward and reverse
primers, respectively (Table S1). The PCR product was digested with
ApaI and Not I, and then inserted into the same site of pBluescript IIKS+ , yielding pBS(ypt71). pBS(ypt71) was digested with Sal I and MluI,
filled in, and ligated to a BamHI linker, yielding pBS(ypt71::BamHI). This
was digested with BamHI and the 1.8-kb ura4+ fragment was inserted
into the same sites, yielding pBS(ypt71::ura4) (Figure 1C). A 4.2-kb XbaI
fragment containing the disrupted ypt71 allele (ypt71::ura4+ ) was used to
transform strain TN29. Disruption was confirmed by southern hybridization
of genomic DNA.
Construction of a strain expressing GFP-tagged
Ypt7 or Ypt71
A GFP-ypt7 fragment including the ypt7 promoter and terminator regions
was amplified by PCR using pZK23 (pBS-ypt7promoter -GFP-ypt7)(11) as
template and gfpypt7-ps and gfpypt7 -tas as forward and reverse primers,
respectively (Table S1). The GFP-ypt7 fragment was introduced into
strain KJ100-7BY (ypt7) from which Ura− transformants were obtained.
This integrant strain (ZK131) harbors a single copy of GFP-ypt7 at the
ypt7 locus. GFP-ypt71 was constructed by 2-step PCR. The ypt71 ORF
921
Kashiwazaki et al.
was amplified by PCR using gfpypt71-ntag and ypt71-tas as forward and
reverse primers, respectively (Table S1). The promoter region of ypt71+
was also amplified using gfpypt71-ps and gfpypt71-pas as forward and
reverse primers, respectively (Table S1). The second PCR were performed
using the first PCR products and GFPS65T as template and gfpypt71-ps and
gfpypt71-tas as forward and reverse primers, respectively (Table S1). The
GFP-ypt71 fragment was introduced into the ZK58. Ura− transformants
were obtained. This integrant strain (ZK116) harbors a single copy of
GFP-ypt71 at the ypt71 locus.
Site directed mutagenesis
Plasmid pREP1(ypt71T22N) was constructed as follows: The N-terminal
region of ypt71T22N ORF was amplified by PCR using ypt71-s and
ypt71T22N-mas as forward and reverse primers, respectively (Table
S1). The C-terminal region of the ypt7T22N ORF was amplified by
PCR using ypt71T22N-ms and ypt71-as as forward and reverse primers,
respectively (Table S1). The PCR product was digested with BamHI and
Not I, and then inserted into BamHI- and Not II-digested pREP1, yielding
pREP1(ypt71T22N). All the mutants were constructed in a similar manner
using appropriate primers (Table S1).
Construction of chimeras
Chimeric molecules were constructed by overlapping PCR(40,41). As
an example, to construct the chimera encoding a fusion of the Ypt7 Nterminal half to the Ypt71 C-terminal half [Ypt7(1–110)-71], the 3 half of the
ypt71+ gene were first amplified with Pfu polymerase (Fermentas) using
primers ypt7(110)-71-cs and ypt7-71-as (Table S2 for primer sequences).
Amplification was for 30 cycles of 30 seconds 94◦ C, 30 seconds 48◦ C
and 3 min 68◦ C. The ypt7(110)-71-cs primer has the ypt7 sequence at
its 5 end and the ypt71 sequence at its 3 end. The products of this
amplification were purified using an Ultrafree-MC (Millipore) and then
included in a second PCR reaction using ypt7 + gene as a template and
ypt7-71-s and ypt7-71-as primers. These outside primers introduce a Sal I
site one nucleotide upstream of the start codon and a Sac I site 500 bp
downstream of the stop codon of the chimeric gene. The PCR product was
purified, digested with Sal I and Sac I, and cloned into similarly digested
pREP1 or pREP41-GFP. All the chimeras were constructed in a similar
manner, using appropriate primers and templates (Table S2).
Staining of vacuolar membranes
Vacuolar membranes were stained with FM4-64 [N(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium-dibromide]
(Molecular Probes) according to Morishita et al.(32) with minor modifications. Cells were harvested, resuspended in 0.5 mL of YE or MM
medium containing 0.5 μL of 8 μM FM4-64 in dimethyl sulfoxide, and then
were incubated with shaking at room temperature for 30 min. Stained
cells were chased with YE or MM medium for 30 min. Vacuole fusion
was induced in water for 60 min. Stained cells were observed under
a fluorescence microscope (model BX51, Olympus) and images were
obtained using a Cool SNAP CCD (charge-coupled device) camera (Roper
Scientific).
Western blotting
ZK134 (GFP-ypt7 ) and ZK130 (GFP-ypt71) were cultured in liquid medium
(YE) for 16 h. Culture aliquots were collected, and crude cell extracts were
prepared as described by Masai et al.(42). Polypeptides were resolved by
SDS-PAGE on 10% gels and then transferred onto polyvinylidene difluoride
membranes (Millipore). Filters were probed with a mouse anti-GFP
antibody (Roche, Basel, Switzerland) at a 1:1000 dilution. Blots were also
probed with a rabbit anti-Spo14 antibody(43), to normalize protein loading.
Immunoreactive bands of GFP fusion proteins were visualized by staining
with an enhanced chemiluminescence (ECL) horseradish peroxidaseconjugated sheep anti-mouse immunoglobulin (IgG) (GE Healthcare)
and ECL-plus detection reagent (GE Healthcare). Immunoreactive bands
of Spo14 were visualized by staining with a horseradish peroxidaseconjugated goat anti-rabbit IgG (Bio-Rad) and ECL detection reagent (GE
Healthcare).
922
Subcellular fractionation
ZK134 (GFP-ypt7 ) and ZK130 (GFP-ypt71) were cultured in liquid medium
(YE) for 16 h. Half of the cells were collected and replaced with water for
3 h. Cells were harvested and resuspended in TES (20 mM Tris-HCl (pH
7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl). After
the addition of phenylmethylsulfonyl fluoride (PMSF), (Tris EDTA saline)
cells were ruptured with glass beads. The lysate was centrifuged at 800 x
g for 5 min to remove cell debris. The supernatant was further centrifuged
at 100 000 x g for 1 h to separate the soluble fraction, the pellet being
resuspended in an equal volume of TES buffer. The samples from each
fraction were subjected to western blot analysis using either the mouse
anti-GFP (Roche) or the rabbit anti-Spo14(43) antibody.
Preparation of recombinant proteins and GTP
overlay assay
Plasmid pGEX(ypt71) was constructed as follows. The ypt71+ cDNA
was amplified by PCR using ypt71gst-ntag and ypt71-as as forward
and reverse primers, respectively. The PCR product was digested with
BamHI and Not I, and then inserted into BamHI- and Not I-digested pGEXKG (GE Healthcare), yielding pGEX(ypt71). Plasmids pGEX(ypt71T22N),
pGEX(ypt71Q67L) and pGEX(ypt71D127A) were constructed similarly.
These plasmids were then used to transform Escherichia coli (E. coli )
Rosetta (Novagen). E. coli strains harboring GST fusion protein constructs
were grown in Luria-Bertani broth (LB) at 37◦ C for 2 h. Protein expression
was induced by the addition of isopropyl β -D-thiogalactoside to 0.1 mM,
and cultures were incubated at 25◦ C for 7 h. Cells from 50 mL of culture
were harvested, washed with binding buffer [PBS buffer containing 1 mM
phenylmethylsulphonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT)]
and suspended in 5 mL of binding buffer. Cells were then ruptured by
sonication at 4◦ C. Lysates were centrifuged at 10 000 x g for 30 min at
4◦ C. Supernatants were added to glutathione-sepharose (GE Healthcare),
which was equilibrated with binding buffer, and incubated at room
temperature for 20 min. Beads were washed with binding buffer three
times. The GTP overlay assay was performed as described by Lapetina
and Reep (1987), Bucci et al.(1992) and Huber et al.(1993) (44–46). GSTfusion proteins were separated by SDS-PAGE. The gel was then soaked in
50 mM Tris-HCl (pH7.5) and 20% glycerol for 30 min and electrophoretically
transferred onto Hybond-ECL nitrocellulose filter (GE Healthcare) in 10 mM
NaHCO3 /3 mM Na2 CO3 (pH9.8). After the transfer, the filter was rinsed
twice for 10 min in GTP-binding buffer (50 mM NaH2 PO4 , pH 7.5, 5 mM
MgCl2 , 0.3% Tween 20, 2 mM DTT. 4 μM ATP as competing substrate),
and then incubated with 1 μCi/mL (37 kBq/mL) α -[32 P]-GTP for 2 h. After
six 5-min washes, the filter was visualized using imaging plate BAS-SR
2025 (Fujifilm) and FLA3000 (Fujifilm).
Acknowledgments
We thank A. Terakita and coworkers of Osaka City University for useful
discussions and M. Yoshida of RIKEN for S. pombe strains. Some of
the S . pombe strains were provided by the Yeast Genetic Resource
Center Japan supported by the National BioResource Project (YGRC/NBRP;
http://yeast.lab.nig.ac.jp/nig/). The present study was supported in part by
Grant-in-Aid for Scientific Research on Priority Areas ‘Genome Biology’ to
C.S., ‘Cell Cycle Control’ and ‘Life of Proteins’ to T.N. from the Ministry
of Education, Culture, Sports, Science and Technology of Japan. J.K. is a
recipient of the Research Fellowship for Young Scientist from the Japan
Society for the Promotion of Science (JSPS).
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Figure S1: Comparison of the amino acid sequences of Ypt7 and Ypt71
orthologs. Sp, Schizosaccharomyces pombe; So, Schizosaccharomyces
Traffic 2009; 10: 912–924
Two Rab7 Homologs in S. pombe
octosporus; Sj, Schizosaccharomyces japonicus. Dark and light shaded
boxes indicate identical and similar residues in four of six proteins,
respectively. Conserved specific residues of Ypt7 and Ypt71 are indicated
by asterisks.
Figure S2: Ypt7, Ypt71 and all Ypt71 mutant proteins can bind to the
HOPS complex. The top three images are immunoblots of GST pull-down
assays using an anti-FLAG antibody (see Materials and Methods). The
bottom image shows a CBB-stained SDS-PAGE gel of purified recombinant
proteins. Asterisks indicate the degraded products under GST-Ypt7(47) and
GST-Ypt71D127A .
Table S1: Primers used in this study
Table S2: Construction of chimeras: primers and templates
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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