41NISR Research GRANT
2010 Research Grant

The mechanism for vacuolar amino acid compartmentalization and its regulation in the budding yeast Saccharomyces
cerevisiae
Takayuki SEKITO
Faculty of Agriculture, Ehime University

Research aims

Results

The vacuole is an organelle, which serves as a storage
compartment of ions, secondary metabolites, and toxic substances as well as a digestive compartment of macromolecules such as proteins. Vacuoles in Saccharomyces cerevisiae contain more than 50% of cellular amino acids. Thisraises the possibility that vacuoles play some importantroles in amino acid metabolism. Recent studies are particularly focusing on the role in autophagic protein degradationinduced under nitrogen starvation. Amino acids generated
in vacuoles by autophagy, have been thought to be recycled
for protein synthesis. However, those export machineries
from vacuoles have not been understood well. In this study,
to elucidate the mechanism of formation and regulation of
vacuolar amino acid pool, we investigated the in vivo function of Avt3 and Avt4, amino acid transporters involved in
the export of glutamine, tyrosine and isoleucine from vacuoles. In addition, we carried out a comprehensive analysis
to identify transporters that localize to the vacuolar membrane. Vacuolar amino acid amounts were measured for themutants of newly identified vacuolar transporters.

1. Effect of AVT3 and/or AVT4 disruption on vacuolaramino acid composition
In vitro analysis using isolated vacuolar membrane vesicles identified several vacuolar amino acid transporters inthe budding yeast S. cerevisiae3–5). Avt3 and Avt4 have beensuggested to extrude glutamine, tyrosine and isoleucine
from vacuoles in a redundant manner3). To examine their invivo functions, vacuolar amino acid compositions werecompared among wild-type, avt3D, avt4D, and avt3Davt4Dcells (Figure 1A). Consistent with previous report, in
avt3Davt4D vacuoles, amounts of glutamine, tyrosine andisoleucine increased. In addition, it is found that most of theother neutral amino acids, including threonine, serine, asparagine, glycine, alanine, valine, methionine, leucine, andproline, also increased. Either single disruption of AVT3 or
AVT4 gene resulted in a minor change of these amino acidamounts. Levels of acidic and basic amino acids were littlechanged by either single or double gene disruption. Thesesuggest that Avt3 and Avt4 export a variety of neutralamino acids from vacuoles in a redundant manner. We assumed that the extrusion of vacuolar amino acids by Avt3
and Avt4 contributes to amino acid recycling during autophagy. In agreement with this, under nitrogen-starved
condition, which induces autophagy, avt3Davt4D vacuoles
contained more neutral amino acids than wild-type vacuoles (Figure 1B). Basic amino acids, which are abundantin vacuoles in nutrient-rich condition, were greatly reduced
in wild-type and avt3D vacuoles in response to nitrogenstarvation (compare Figure 1A and B). On the other hand,more than 20 fold of basic amino acids was retained in
avt4D and avt3Davt4D vacuoles when compared with wildtype vacuoles (Figure 1B). These suggest that both Avt3
and Avt4 are involved in the recycling of neutral aminoacids during autophagy. In addition, Avt4 may have a distinct function from Avt3, which is the export of basic aminoacids from vacuoles.

Methods
Amino acid composition analysis
The budding yeast S. cerevisiae cells were cultured inYPD (1% yeast extract, 2% polypeptone, 2% dextrose) toearly logarithmic phase. For nitrogen-starvation, logarithmically growing cells in YPD were washed and suspended
with SD-N (0.17% yeast nitrogen base w/o amino acids andammonium sulfate, 2% dextrose), and further cultured for 6hours. Cells were collected and subjected to cupric ion
treatment to extract vacuolar amino acid pool as describedpreviously1,2). Amino acid amounts were measured by using
Hitachi L-8800 automatic amino acid analyzer.
Microscopic analysis
GFP fluorescence was observed by using Olympus IX71
fluorescent microscope equipped with Metamorph imagingsoftware.

2.

Function of N-terminal hydrophilic region of Avt4
In predicted topology models, Avt4 possesses a long Nterminal hydrophilic region, of which length is about 300
amino acids. In some cases, such long hydrophilic region oftransporter is post-translationally modified and/or interacts

42Figure 1 Vacuolar amino acid amounts in cells cultured in nutrient-rich (A) and in nitrogen-starved (B) conditions. Wild-type (white bar), avt3D (lightgray bar), avt4D (dark gray bar), and avt3Davt4D (black bar) cells were cultured and treated with cupric ion to extract those vacuolar amino acid pools as described in “Methods”. The amounts of basic amino acids, which were abundant in vacuoles, were presented in separate graphs (right).

Figure 2 Effect of the truncation of N-terminal hydrophilic region from Avt4 on vacuolar amino acid amounts under nitrogen starvation. Vacuolar aminoacid amounts in avt3D cells carrying empty plasmid (white bar) and avt3Davt4D cells carrying either empty plasmid (light gray bar) or plasmid encoding
GFP-Avt41-713 (dark gray bar), GFP-Avt4101-713 (black bar), or GFP-Avt4201-713 (hatched bar) were measured as in Figure 1B.

with the other protein to regulate its transport activity. To
elucidate the function of the N-terminal region of Avt4,
GFP-fused truncated Avt4 variants, GFP-Avt4101-713, GFPAvt4201-713, and GFP-Avt4301-713, in which the N-terminal100, 200, and 300 amino acids were deleted, respectively,were expressed in avt3Davt4D cells, and examined for theircellular level and subcellular localization under nitrogenstarvation. Western blot analysis and microscopic observation revealed that, the level of GFP-Avt4301-713 was muchless than that of full-length Avt4 (GFP-Avt41-713). In addition, vacuolar membrane localization of GFP-Avt4301-713
was defective when compared with GFP-Avt41-713. On the43

other hand, GFP-Avt4101-713 and GFP-Avt4201-713 exhibited
similar cellular levels to that of GFP-Avt41-713 and no defectin the vacuolar membrane localization. Next, we comparedvacuolar amino acid amounts in avt3Davt4D cells expressing GFP-Avt4101-713 or GFP-Avt4201-713 with GFP-Avt41-713
under nitrogen starvation. As shown in Figure 2, vacuolaramino acid amounts in cells expressing either truncatedAvt4 were more than 2-fold of those in cells expressing
GFP-Avt41-713. This implicates that the N-terminal region ofAvt4 plays an important role in amino acid export fromvacuoles.
3.

Identification of vacuolar transporters
Thirty putative transporters encoded in S. cerevisiae
genome, which have not been characterized, were fused toGFP at either N- or C-terminal end and examined for thesubcellular localization by fluorescent microscope. Amongthem, six transporters were found to localize to the vacuolar
membrane. However, disruption of each gene did notchange the vacuolar amino acid content. It is possible thatthe defect caused by the disruption of each transporter gene
was compensated by overlapping function of the othertransporters. Amino acid composition analysis is currently
in progress for strains with multiple gene disruptions, inwhich some of the newly identified six transporter genes together with other transporter genes, including AVT genes,
were disrupted, as well as for strains overexpressing each of
the six transporters.
Conclusion
Amino acid composition analysis suggested that both
Avt3 and Avt4 extrude variety of neutral amino acids fromvacuoles. Furthermore, we obtained the data suggesting that
Avt4 is involved in the export of basic amino acids fromvacuoles during nitrogen starvation. Results of amino acidtransport assay using isolated vacuolar membrane vesicles
were consistent with the suggested broad substrate specificity of Avt3 and Avt4 (data not shown). We speculate thatthe vacuolar amino acid transport system may participate in
the maintenance of cellular amino acids at optimal level ina concerted manner with other systems, such as amino acidtransporters at the plasma membrane and enzymes involvedin amino acid metabolism. Considering available data, it islikely that activity of vacuolar amino acid transporters isregulated in response to change in the cellular amino acidlevel. In this study, we found that cells expressing truncatedAvt4, in which the N-terminal hydrophilic region wasdeleted, retained more amino acids in vacuoles than cells
expressing full-length Avt4 during nitrogen starvation.
Since the cellular level and the vacuolar membrane localization were not affected by the truncation, this result suggests that the N-terminal region is required for the aminoacid transport activity of Avt4. We expect that the N-terminal region of Avt4 is involved in the regulation of activity.
This study also revealed that six uncharacterized putativetransporters were located at the vacuolar membrane. Microarray analysis suggested that some of them were inducedby nitrogen/amino acid starvation. This implies the close
relation of these transporters to amino acid metabolism.
Further investigation is in progress.
References1)

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