Novel families of vacuolar amino acid transporters

Authors


Abstract

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. © 2008 IUBMB IUBMB Life, 60(8): 519–525, 2008

INTRODUCTION

The cytosolic amino acid concentration must be maintained at a relatively constant level for effective protein synthesis in all living organisms. Loss of this maintenance of amino acid level results in damage to cellular metabolism and survivability. The regulatory mechanism for amino acid homeostasis has been mainly studied in the budding yeast Saccharomyces cerevisiae because of its strong genetics background. So far, the results of such studies have argued for the accumulation of amino acids from the external environment and biosynthesis1–4. In addition to these sources, however, we should pay attention to the role of the organellar compartmentalization of amino acids. In S. cerevisiae, several amino acids, especially basic amino acids, are highly concentrated in the vacuoles. In contrast, glutamate and aspartate are exclusively localized in the cytosol5. More than 90% of arginine is compartmentalized in the vacuoles, whereas only 11% of aspartate is in them6. This implies that the vacuole is the selective compartment for regulating the cytosolic level of amino acids. It has been difficult to assess the significance of vacuolar amino acid compartmentalization on the amino acid homeostasis in the cytosol since the pathways for transporting amino acids across the vacuolar membrane are not well understood at the molecular level. Recent studies using S. cerevisiae revealed the existence of vacuolar amino acid transporters in the amino acid/auxin permease family (AAAP) and the major facilitator superfamily (MFS)7–10 and thus open the way to assess the significance of vacuolar amino acid compartmentalization at the molecular level. In this manuscript, we briefly characterize vacuolar amino acid transporters in S. cerevisiae as well as the homologous genes in Arabidopsis thaliana and mammals. The physiology of some transporters in response to nitrogen starvation is also discussed.

COMPARTMENTALIZATION OF AMINO ACIDS IN VACUOLES

Of the large amount of amino acids taken up into fungal cells, only a small portion is involved in metabolism and protein synthesis. The rest is metabolically inert11–13. It is likely that amino acids are compartmentalized to separate these two metabolically distinct pools. A study using the yeast Candida utilis demonstrated that vacuoles play a key role in this compartmentalization. The bulk of the amino acids originating from the vacuoles were labeled only slowly by radioactive tracers, while only a small portion of the total amino acid pool from the cytosol was labeled very rapidly14. Subsequent analysis enabled by the development of vacuole purification methods from S. cerevisiae estimated that 60% of the total cellular amino acids was pooled in vacuoles5. Thus, vacuoles function as a large storage pool of amino acids, while the remaining cytosolic pool is engaged in metabolism and protein synthesis. Importantly, it was found that the amino acid composition differs between the cytosol and vacuoles. The bulk of basic amino acids and glutamine are localized in vacuoles, while glutamate, and aspartate are almost completely excluded from this compartment and localized in the cytosol5. The vacuole is thus the selective compartment for a specific subset of cellular amino acids.

ACTIVE TRANSPORT OF AMINO ACIDS BY VACUOLES

The experiment with isolated yeast vacuoles uncovered an exchange reaction of arginine without an apparent energy source15. Although this implies that amino acid flux occurs across the vacuolar membrane, the mechanism of active transport for amino acids remained to be observed. Active transport of amino acids by yeast vacuoles was initially demonstrated by an experiment using vacuolar membrane vesicles, which are right-side-out oriented and depleted of the endogenous amino acids normally present in vacuoles16. Active transport of arginine by the vacuolar membrane vesicles was driven by an electrochemical gradient of protons across the vacuolar membrane generated via the action of proton pumping vacuolar ATPase (V-ATPase)16–18. In addition to arginine, nine other amino acids-lysine, histidine, phenylalanine, tryptophan, tyrosine, glutamine, asparagine, isoleucine, and leucine-were also shown to be taken up actively into the vacuolar membrane vesicles (Fig. 1)19. At the steady-state level of uptake, more than fivefold concentration gradients of all of these amino acids except for asparagine were established; asparagine uptake was less efficient, establishing a concentration gradient of about twofold. The transport systems of the vacuolar membranes have lower affinities for substrates: the Kt values are mostly in the order of 10−3 M19, which are 1–2 orders of magnitude higher than those of the transport systems of the plasma membranes. The kinetics studies for these transport activities predicted the presence of seven independent transport systems in the vacuolar membrane: those for arginine, arginine-lysine, histidine, phenylalanine-tryptophan, tyrosine, glutamine-asparagine, and isoleucine-leucine, all of which are driven by an electrochemical gradient of protons, representing H+/amino acid antiporters19. In addition, it was found that arginine uptake was markedly stimulated by histidine due to an exchange transport of arginine outside with histidine inside the vacuolar membranes20.

Figure 1.

Vacuolar amino acid transporters in S. cerevisiae. Ten amino acids (Arg, Lys, His, Phe, Trp, Tyr, Gln, Asn, Ile and Leu) are actively transported into vacuole, whereas acidic amino acids (Glu and Asp) are exclusively localized in cytosol. Amino acid transporters responsible for the vacuolar transport of these, though not all, amino acids are identified. Most of them couple amino acid transport with protons generated by the vacuolar H+-ATPase. Transporters for GABA (Uga4) and cystine (Ers1) are also identified and are possibly an antiporter and a symporter with protons, respectively.

Interestingly, Ohsumi et al.21 developed a cupric ion treatment method to disrupt the permeability barrier for ions and small-sized metabolites of the plasma membrane, but not the vacuolar membrane. This method enables the examination of the vacuolar amino acid pool with intact cells. Kitamoto et al.6 examined the amino acid composition of the cytosolic and vacuolar fractions of cupric ion-treated cells cultured in media supplemented with various amino acids, and obtained results that are in parallel with those previously obtained by vacuolar membrane vesicles.

By contrast to yeast, relatively little is known about the vacuolar transport of amino acids in the other organisms. Uptake of a subset of amino acids was extensively studied by isolated vacuoles of mesophyll cells from barley22–24, but the active transport of amino acids depending upon ATP or the electrochemical gradient of protons across the vacuolar membrane has not been observed in barley vacuoles25. Vacuoles have long been considered to be storage compartments; yet because of their large volume, vacuolar amino acid concentrations are estimated to be lower than cytosolic ones26–28. Vacuolar amino acid contents vary under growth conditions that affect nitrogen remobilization. In oilseed rape (Brassica napus L.), high nitrogen supply led to the accumulation of amino acids into vacuoles in young leaves but not in mature or old leaves, suggesting that excess nitrogen stored as amino acids is utilized during leaf development28. Carbohydrate starvation caused autophagic degradation of cytosolic proteins in vacuoles29, resulting in significant accumulation of free amino acids30, 31. These data suggest that amino acids stored in vacuoles may be exported and used as respiratory substrates or for protein synthesis30. So far, the mechanism of amino acid transport across the vacuolar membrane has not been understood in plants.

IDENTIFICATION OF VACUOLAR AMINO ACID TRANSPORTERS

Although the genes responsible for amino acid transport across the vacuolar membrane had long been unidentified, recent studies using a reverse genetics approach successfully identified the genes for vacuolar amino acid transporters in S. cerevisiae, which are listed in Table 1 and summarized in Fig. 1. Plant homologues are listed in Table 2.

Table 1. S. cerevisiae vacuolar amino acid transporters and related proteins
FamilySubfamilyProteinSelectivityVacuolar directionReference
  • *

    Competitive inhibition studies by Sato et al.19 have shown that asparagine and glutamine and leucine and isoleucine utilize the same vacuolar uptake system.

  • Abbreviation for lysosomal cystine transporter family8.

AAAPAVTAvt1Gln, (Asn*), Ile, (Leu*), TyrIn9
Avt2Unknown 9
Avt3Gln, (Asn*), Ile, (Leu*), TyrOut9, 32
Avt4Gln, (Asn*), Ile, (Leu*), TyrOut9, 32
Avt5unknown 9
Avt6Asp, GluOut9
Avt7Unknown 9
MFSVBAVba1His, LysIn10
Vba2His, Arg, LysIn10
Vba3His, LysIn10
Vba4Unknown 10
Vba5Unknown 10
Azr1Unknown 10, 33
Sge1Unknown 10, 34
 Atg22Leu, Tyr, IleOut32
APC Uga4GABAIn35
LCT Ers1CystineOut36
Table 2. Putative vacuolar amino acid transporters identified in A. thaliana
GeneProteinMethod*Reference
  • *

    A, proteome, cell culture; B, proteome, leaf; C, GFP, cell culture.

  • AtCAT8-GFP localized to the plasma membrane and intracellular membranes37.

  • Fujiki and Kakinuma, unpublished.

At1g58030AtCAT2A, C37, 38
At3g03720AtCAT4A, B38, 39
At1g17120AtCAT8A, C37, 38
At1g05940AtCAT9A38
At2g39130AVT1-likeA38
At2g41190AVT1-likeA, C38
At3g28960AVT1-likeA38
At5g65990AVT3-likeC 
At2g40420AVT6-likeA38
At3g30390AVT6-likeA, B38, 39
At5g40670ERS1-likeA38

AVT Family

Russnak et al.9 identified a protein family of vacuolar amino acid transporters from S. cerevisiae. This family, the AVT family, is related to neuronal γ-aminobutyric acid (GABA)-glycine vesicular transporters such as Caenorhabditis elegans UNC-4740 and the vertebrate homologues from rat (VGAT, 40) and mouse (VIAAT, 41), which belong to the amino acid/auxin permease (AAAP) family8. The AVT genes were characterized by a transport experiment with vacuolar membrane vesicles prepared from deletion mutants. It was shown that Avt1 is involved in the vacuolar uptake of glutamine, leucine, isoleucine, asparagine, and tyrosine, whereas both Avt3 and Avt4 function for the vacuolar export of these same amino acids. Avt6 mediates the efflux of glutamate and aspartate. Transport substrates of Avt2, Avt5, and Avt7 remain unknown. LYAAT, a member of the AAAP family from rat, which is closely related to Avt3/Avt4, was found to function as a neutral amino acid exporter at lysosomal membranes42.

Orthologues of AVT transporters have also been found in plants43, 44. In A. thaliana, AVT homologues are categorized into three subfamilies: AtAVT1-, AtAVT3/4-, and AtAVT6-subgroups, which include 10, 4, and 5 genes, respectively. One AtAVT homologue, ANT1 (aromatic and neutral transporter), transports aromatic, and neutral amino acids when expressed in yeast cells45. ANT1-expressing yeast cells also transport auxin, which is structurally related to tryptophan45, but it has not been determined whether auxin is a native substrate of ANT1. Several members of the AtAVT family have been identified by proteomic approaches for isolated vacuoles (Table 2). In addition, ANT1-like AtAVT3/4 genes are functionally competent to mediate amino acid flux in yeast Δavt3Δavt4 mutants (Fujiki et al., unpublished data). Taken together, these data indicate that AtAVT family members may mediate the H+-coupled export/import of amino acids across the vacuolar membrane in a similar manner to yeast. It will be necessary to evaluate their physiological significance using AtAVT-deleted plants.

VBA Family

As described earlier, the bulk of basic amino acids, such as arginine, histidine and lysine, are transported into yeast vacuoles. The genes encoding transporters for these amino acids were identified by Shimazu et al.10. These genes constitute a new family (VBA) in the major facilitator superfamily (MFS) of S. cerevisiae7. The results of experiments with vacuolar membrane vesicles from vba deletion mutants indicate that Vba1 and Vba3 are involved in the uptake of histidine and lysine and Vba2 in the uptake of three basic amino acids. All of these are proton/amino acid antiporters. The VBA family contains four other members (Table 1). Vba4 is also involved in the vacuolar transport of basic amino acids (Kakinuma et al., in preparation). The amino acid sequences of Vba5 and Vba3 are almost identical, differing by only three amino acids over the entire 458 amino acid sequence. Thus, it is plausible that Vba5 is also involved in the transport of basic amino acids across the vacuolar membrane. To understand the significance of the multiplicity in transporters for basic amino acid uptake, it is important to know the details of the substrate specificity, and the regulation of the activities in response to changes in environmental conditions. Azr1 and Sge1 are also members of the VBA family, but their transport activities have not been examined; it has been reported that they are responsible for drug-resistance33, 34.

Vacuolar arginine uptake is also inhibited by deletion of BTN146, which encode a protein similar to human CLN3 (39% identity/59% similarity). Mutations in CLN3 gene cause Batten disease, which is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death47. Btn1 is also required for strict regulation of vacuolar pH48, 49. Btn1/CLN3 is a vacuolar/lysosomal transmembrane protein and the defects in Δbtn1 cells are complemented by transforming CLN346. Although the basis for the vacuolar pH imbalance caused by deleting BTN1 is still not clear, it could greatly affect the activity of the vacuolar transport system which is coupled with proton transport. In addition, Btn1 has no homology to any other yeast transporter proteins. Thus, it is more likely that the defect in arginine transport in Δbtn1 cells is a secondary consequence of a loss of vacuolar pH regulation46.

Other Vacuolar Amino Acid Transporters

In addition to the AVT and VBA families, several other transporters have been strongly suggested to mediate amino acid transport across the vacuolar membrane.

UGA4

Although Avt proteins are closely related to GABA transporters on mammalian synaptic vesicles, GABA transport activity by them has not been detected9. S. cerevisiae is able to utilize GABA as its sole nitrogen source and Uga4 is responsible for the uptake of GABA by cells50. This uptake can be inhibited by bafilomycin A1, a specific inhibitor of vacuolar ATPase35. This suggests that the cellular uptake of GABA involves vacuolar uptake, although the physiological rationale for GABA transport into vacuole has not been elucidated. In addition, Uga4 localizes to vacuoles35. Thus, it is likely that Uga4 is a GABA-specific transporter in the vacuolar membrane. Uga4 belongs to the amino acid-polyamine-choline (APC) family43 that contains amino acid permeases at the plasma membrane, such as the general amino acid permease Gap1 and specific amino acid permeases from S. cerevisiae.

The APC family also contains cationic amino acid transporters (CAT), which have been characterized in mammals and plants as high-affinity transporters for cationic amino acids37, 43. Nine AtCATs were found in the A. thaliana genome and several members are suggested to be localized to the vacuole (Table 2)37, 44, although the biochemical properties have not yet been reported.

ERS1

Yeast Ers1 is similar to the human CTNS gene product cystinosin, a lysosomal membrane protein (28% identity/46% similarity). The CTNS mutation in humans causes a renal tubule disorder known as Fanconi syndrome, which leads to growth retardation, hypothyroidism, photophobias, and neurological dysfunctions if untreated51. Cystinosin is a proton/cystine symporter52. A yeast strain lacking ERS1 is sensitive to hygromycin B. Although the basis for this sensitivity is not understood, it is complemented by the human CTNS gene. In addition, Ers1 localizes to the vacuolar membrane as well as to endosomes36. Although neither cystine transport activity of Ers1 nor a change in vacuolar cystine level in Δers1 cells has been demonstrated, it is very likely that Ers1 is a functional homologue of cystinosin and exports cystine from vacuoles.

ATG22

It has recently been reported by Yang et al.32 that Atg22 is an amino acid transporter for exporting tyrosine/leucine/isoleucine from vacuoles. Atg22 was first identified as a factor required for the breakdown of autophagic bodies that originated from the fusion of autophagosomes with vacuoles53. Under condition of nitrogen starvation, amino acids that result from the vacuolar degradation of bulk protein delivered by autophagy should be recycled to sustain a certain level of protein synthesis. It has been assumed that the export activity of amino acids from vacuoles is essential to achieve this. Atg22 is classified into MFS54 and localizes to the vacuolar membrane. Although the transport activity of Atg22 has not been examined with the vacuolar membrane vesicles, the amounts of vacuolar tyrosine, leucine and isoleucine in Δatg22 cells increased relative to those in wild type cells. Leucine auxotrophic Δatg22 cells failed to maintain their survivability in nitrogen starvation medium (SD-N), whereas supplementing leucine to SD-N resulted in increased survivability. Two other vacuolar amino acid exporters, Avt3 and Avt4, are required in addition to Atg22 for the maintenance of viability in SD-N32.

THE PHYSIOLOGICAL ROLE OF THE VACUOLAR AMINO ACID POOL

Vacuoles are the large-sized organelles involved in the cellular homeostasis of ions and metabolites as well as osmotic regulation through various transporters in response to environmental stress. The amino acid compartmentalization in vacuoles is dynamically linked with such a homeostatic function for the maintenance of the cytosolic amino acid concentration in yeast and plant cells. An elegant work by Kitamoto et al.6 has shown the significance of vacuoles in amino acid homeostasis in yeast. Arginine is a good nitrogen source, and excess arginine in the cytosol is degraded by arginase. However, by supplementing the media with basic amino acids, such as lysine, histidine, and arginine, the vacuolar amino acid pool of S. cerevisiae cells was enlarged while the cytosolic amino acid pool stayed fairly constant. The authors further demonstrated the removal of arginine from the vacuoles during nitrogen starvation. Yeast cells thus may utilize their vacuolar arginine pool as a nitrogen source. Vacuolar compartmentalization of amino acids as a nitrogen supplier has also been demonstrated in a study of autophagy55. Upon nitrogen starvation, the vacuole decomposes the bulk of cytoplasmic proteins delivered by autophagy. Resulting amino acids are recycled from vacuoles to the cytoplasm by vacuolar amino acid exporters. Atg22, Avt3, and Avt4 have been suggested to function in this process as they have been shown to maintain survivability under starvation32.

Although we can evaluate the activity of individual transporters with studies of the net movement of amino acids with vacuolar membrane vesicles, the role of transporters in vacuolar compartmentalization in vivo has not been well understood. Atg22, Avt3, and Avt4 are all required for survivability under nitrogen starvation condition. However, cell growth phenotypes were not observed upon deletion of the other transporters. This could be due to functional redundancy among the multiplicity of transporters, including unknown ones, with similar substrate specificity. Thus, to understand the physiological role of vacuolar amino acid compartmentalization, further identification of vacuolar amino acid transporters is required. Special attention should be paid to diversity in the expression of these multiple genes in response to various culture conditions. Even in perturbed conditions, such as the absence of amino acids or the presence of excess amino acids, the regulation of amino acid permease activity at the plasma membrane and/or amino acid biosynthesis activity could be enough to maintain amino acid homeostasis. To define the role of vacuolar amino acid transporters, the relationships to these activities also have to be considered. Indeed, intact cells carrying vba mutations exhibit a reduction of histidine uptake10, indicating that vacuolar transport activity for amino acids may affect the cellular uptake activity. In any case, further investigation is required to uncover the details of amino acid homeostasis machinery and the importance of vacuolar amino acid compartmentalization.

Acknowledgements

This work was in part supported by a Grant-in-Aid for Scientific Research (to T.S. and Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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