38

Noda Institute for Scientific Research GRANT
2013 Research Grant

Molecular machinery and regulatory system of vacuolaramino acid compartmentalization as a new target for yeast
engineering
Yoshimi Kakinuma
Faculty of Agriculture, Ehime University

Research aims
The vacuole, which is a membrane-bound organelle present in all eukaryotic microorganisms and plants, serves as acompartment for the storage of metabolic products and sequestration of xenobiotics. It also functions as a digestive
compartment for macromolecules such as proteins, which isa common feature with lysosomes in mammalian cells. In
recent years, the digestive role of vacuoles has attracted increasing attention, specifically, the process of autophagy
that is induced during nitrogen starvation. More than 50%of the cellular amino acids are found in the vacuoles. Although it is plausible that vacuoles play a critical role inamino acid metabolism, including protein synthesis anddegradation, the molecular mechanism of vacuolar aminoacid compartmentalization and regulation, and its association with amino acid metabolism remain unclear. In thisstudy, we used the budding yeast Saccharomyces cerevisiae
as a model organism to establish the molecular basis ofvacuolar amino acid compartmentalization for applicationin the fermentation industry. We also analyzed the regulatory mechanism of the vacuolar amino acid transporter
Avt4 and identified novel vacuolar amino acid transporters.
Methods
Strains and growth conditions
The derivatives of S. cerevisiae strain X2180-1B (MATαSUC2 mal mel gal2 CUP1) were used in this study. The
cells were cultured in SD+CA (0.17% yeast nitrogen basew/o amino acids and ammonium sulfate, 0.5% casaminoacids, 0.5% ammonium sulfate, and 2% dextrose) to theearly logarithmic phase. For sporulation, the cells werewashed two times, suspended in sporulation medium (0.1%
yeast extract, 0.05% glucose, and 1% potassium acetate),and then cultured at 25°C for 6 days.
Analysis of amino acid composition
The cells were collected and subjected to cupric ion
treatment to extract the vacuolar amino acid pool, as described previously.1) The amount of the amino acids wasmeasured using Hitachi L-8900 automatic amino acid analyzer.

Amino acid transport assay using vacuolar membranevesicles
Vacuolar membrane vesicles were prepared by the Ficoll
floating method, as previously described.2) The efflux ofamino acids from vesicles was measured according to theprevious report.3) Briefly, the reaction was initiated by adding 2 mM ATP to vesicles preloaded with 14C-labeledamino acids and then terminated by diluting with ice-coldbuffer. Radioactivity of the vesicles recovered by vacuum
filtration on cellulose acetate membrane filters was determined using a liquid scintillation counter. ATP-dependent
uptake of amino acids and calcium was assayed as described previously.2, 4)
Results
1. Regulation of vacuolar amino acid transport activity by the N-terminal hydrophilic region of Avt4
Avt4 mediates the extrusion of various neutral aminoacids from the vacuoles in a redundant manner with its
closest homologue Avt3. In addition, Avt4 is involved in theextrusion of basic amino acids that are highly accumulated
in the vacuoles.3, 5) When cells are subjected to conditionsof nitrogen starvation, the content of vacuolar basic aminoacids is rapidly reduced. Since this reduction is significantly suppressed in avt4∆ cells, it is likely that the vacuolar amino acid extrusion by Avt4 increases under these conditions.3) The hydrophilic region of transporter proteins isknown to regulate their activity. Topology prediction ofAvt4 suggests the presence of a long hydrophilic region at
the N-terminus (Fig. 1A). Fungal homologues of Avt4 also
possess a similar long hydrophilic region and an alignmentof the two proteins reveals five conserved motifs (motifI-V) in this region (Fig. 1A). The expression level and vacuolar membrane localization of Avt4 mutants deleted foreach of the five motifs (Avt4∆I-∆V) and expressed under
the native AVT4 promoter in avt3∆avt4∆ cells were almost
the same as the wild-type Avt4. The content of neutralamino acids in the vacuoles was increased by the expression of Avt4∆I, ∆II, ∆III, or ∆IV, but decreased by the expression of Avt4∆V, compared to that in the vacuoles expressing wild-type Avt4 (Fig. 1B). The content of basic
amino acids in the vacuoles was also highly reduced byAvt4∆V expression (Fig. 1B). In contrast, the content of		39Fig. 1. Functional analysis of the N-terminal hydrophilic region of Avt4.
(A) Predicted topology and the conserved motifs (I-V) in the N-terminalhydrophilic region of Avt4. (B) Changes in the alanine transport activity ofvacuolar membrane vesicles by the deletion of the conserved motif. 14Clabeled alanine was preloaded into vacuolar membrane vesicles isolated
from avt3∆avt4∆ cells carrying an empty vector (closed circles) or a vector expressing wild-type Avt4 (open circles), Avt4∆IV (open triangles), orAvt4∆V (open squares), and further incubated in the presence (solid lines)
or absence of ATP (dotted lines). The amount of preloaded 14C-labeled alanine (0 min) was considered to be 100%. The relative amount of alanine in
the vesicles at each time point was plotted. The results are expressed as
mean±SD of three independent experiments. (C) Vacuolar amino acid content in avt3∆avt4∆ cells expressing the Avt4 mutant deleted for the conserved motif. Vacuolar amino acids extracted from the indicated strains
were analyzed with an amino acid analyzer. The results are expressed as
mean±SD of three independent experiments.

acidic amino acids such as glutamate and aspartate remained unchanged. Following the addition of ATP, a slightefflux of alanine from the vacuolar membrane vesicles isolated from avt3∆avt4∆ cells expressing wild-type Avt4 wasobserved and was further accelerated by the expression ofAvt4∆V (Fig. 1C). In contrast, alanine was taken up into
the vesicles expressing Avt4∆IV as well as vesicles isolated
from avt3∆avt4∆ cells carrying an empty vector (Fig. 1C).
These results demonstrate that the N-terminal hydrophilic
region of Avt4 regulates vacuolar amino acid extrusion both
positively and negatively.
2. Identification and characterization of novel vacuolar amino acid transporters6, 7)
The S. cerevisiae genome encodes for seven members of
the AVT transporter family. Among them, Avt1, Avt3, Avt4,and Avt6 are known to be involved in vacuolar amino acidtransport,5) while Avt2, Avt5, and Avt7 are uncharacterized.
We found that GFP-fused Avt7 localizes to the vacuolar
membrane. To explore the possibility of its involvement invacuolar amino acid transport, we examined the effect of
AVT7 disruption on vacuolar amino acid content. When
AVT7 was disrupted in avt3∆avt4∆ strain, in which vacuolar amino acid extrusion is minimized, the content of various neutral amino acids in the vacuoles was found to increase. Among them, the increase in glutamine and proline
was remarkable. In addition, the ATP-dependent uptake ofthese amino acids into vacuolar membrane vesicles isolated
from avt3∆avt4∆avt7∆ cells also increased, when compared with vesicles from avt3∆avt4∆ cells (Fig. 2A). On

Fig. 2. Effect of AVT7 expression on the net uptake of amino acids byvacuolar membrane vesicles and sporulation efficiency.
(A) Vacuolar membrane vesicles were prepared from the avt3∆avt4∆/
empty vector (open circles), avt3∆avt4∆avt7∆/empty vector (closed circles), and avt3∆avt4∆avt7∆/pGPD-AVT7 (closed squares). [14C]glutamine(left) or [14C]proline (right) were preloaded into the vacuolar membranevesicles. After the addition of 2 mM ATP, the vesicles were filteredthrough a cellulose acetate filter (pore, 0.45 μm) at the indicated time
points and the radioactivity remaining on the filter was determined using aliquid scintillation counter. The net uptake activity was calculated by subtracting the amount of amino acids in the preloaded vesicles (0 min) fromthat at each time point. (B) After culturing the diploid cells in sporulation
medium for 6 d, the total number of cells and asci was counted under a microscope to calculate the sporulation efficiency. The results are represented
as mean±SD of five independent experiments. *, P=0.019; **, P=0.049;
and ***, P=0.0013.

Fig. 3. ATP-dependent uptake of amino acids and calcium by vacuolar
membrane vesicles.
Uptake assays were performed for the indicated amino acids and calcium
into the vacuolar membrane vesicles isolated from wild-type (open circles) and ypq1∆ (open triangles) cells. The results are represented as
mean±SD of three independent experiments.

the other hand, overexpression of AVT7 resulted in decreased uptake of glutamine and proline into vacuolar
membrane vesicles (Fig. 2A). These results suggest that
Avt7 is a novel vacuolar amino acid transporter, which isinvolved in the extrusion of neutral amino acids from vacuoles. The recycling of vacuolar amino acids is particularly
important for protein synthesis under conditions of nitrogenstarvation. The content of various neutral amino acids inthe vacuoles of avt3∆avt4∆avt7∆ cells was considerably
higher than in those of avt3∆avt4∆ cells in this condition.
The efficiency of sporulation, which is induced by nitrogenstarvation, was slightly reduced in avt7∆ as well as
avt3∆avt4∆ diploid cells, and was significantly decreasedby avt3∆avt4∆avt7∆ triple disruption (Fig. 2B). These data
demonstrate the involvement of vacuolar amino acid recycling in the adaptation to nutrient starvation.
Ypq1, the S. cerevisiae homologue of lysosomal basic
amino acid transporter LAAT-1/PQLC2 in nematode/rat, localizes to the vacuolar membrane.8) We compared the ATPdependent uptake of basic amino acids such as lysine, arginine, and histidine into the vacuolar membrane vesicles isolated from wild-type and ypq1∆ cells. YPQ1 disruption drastically decreased lysine uptake, partially impaired arginine
uptake, but did not largely affect histidine uptake (Fig. 3).

40The uptake of tyrosine and calcium remained unaffected in
the vesicles of ypq1∆ cells (Fig. 1). In addition, the vacuolar
uptake of basic amino acids depends on the proton motive
force generated by V-ATPase.2) To assess the effect of YPQ1
disruption on the V-ATPase activity, vacuolar membrane
vesicles were subjected to Western blot analysis for VATPase subunit Vph1. Both the wild-type and ypq1∆ vesicles exhibited similar expression of Vph1 (data not shown).
Measurement of the quenching of quinacrine fluorescenceshowed that a pH gradient was generated in ypq1∆ as wellas wild-type vesicles (data not shown). Thus, YPQ1 disruption does not appear to affect the V-ATPase activity. These
results suggest that Ypq1 is specifically involved in the vacuolar uptake of lysine and arginine.

crosstalk between vacuolar amino acid transport and aminoacid metabolism and to understand the physiological importance of the vacuolar amino acid pool.
References
1)
2)
3)

4)

Conclusions
This study demonstrates that, among the five conserved
motifs found in the N-terminal hydrophilic region of Avt4,motifs I, II, III, and IV facilitate the vacuolar extrusion ofamino acids, whereas motif V represses it. It is likely thatthe amino acid transport activity is regulated by Avt4,
thereby providing the first insight into the regulatory mechanism of vacuolar amino acid transport. Our results also
show that Avt7, a member of the AVT transporter family,
mediates the extrusion of various neutral amino acids fromvacuoles. The amino acids extruded by Avt7, Avt3, andAvt4 are probably utilized for the process of sporulation
during nitrogen starvation. In addition, Ypq1, a homologueof the nematode/rat lysosomal amino acid transporter, participates in the uptake of lysine and arginine into the vacuoles. Further research is required to identify and explore theregulation of vacuolar amino acid transporters to reveal the5)
6)

7)

8)

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