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Human Molecular Genetics Advance Access originally published online on March 6, 2007
Human Molecular Genetics 2007 16(9):1007-1016; doi:10.1093/hmg/ddm046
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells

Seasson Phillips Vitiello1, Devin M. Wolfe1 and David A. Pearce1,2,3,*

1 Center for Aging and Developmental Biology, Aab Institute of Biomedical Sciences, 2 Department of Biochemistry and Biophysics and 3 Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA

* To whom correspondence should be addressed at: Center for Aging and Developmental Biology, Box 645, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Fax: +1 5855061972; Email: david_pearce{at}urmc.rochester.edu

Received December 22, 2006; Accepted February 26, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-{Delta}), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-{Delta}. Using the yeast model for the Batten disease, we have determined that although btn1-{Delta} results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-{Delta}), in combination with btn1-{Delta}, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-{Delta}, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-{Delta} cells is detrimental and is suggestive that btn1-{Delta} and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a neurodegenerative disorder resulting in vision loss, cognitive and motor decline, dementia, increased occurrences of untreatable seizures and eventual death in the second or third decade of life. The disease is characterized pathologically by accumulation of hydrophobic lipofuscin-like material called ceroid in the lysosomes. JNCL patients harbor recessive mutations in the CLN3 gene, which encodes a predicted endosomal/lysosomal transmembrane protein (reviewed in 1). Understanding CLN3 function is crucial to understanding the disease and identifying potential therapeutics.

The amino acid sequences of the budding yeast Btn1p and human CLN3 are 39% similar and 59% identical (2,3). Moreover, plasmid-derived expression of CLN3 can complement for the absence of Btn1p (btn1-{Delta}), indicating that Btn1p is a functional homolog of CLN3 (4,5). Btn1p has been shown to localize to the vacuole and has been implicated in several cellular pathways (6,7). Btn1p may be involved in regulating pH balance, as btn1-{Delta} cells have elevated plasma-membrane-ATPase activity that results in increased acidification of the growth media. Furthermore, there is a decreased vacuolar pH in the early log phase, which continues to rise throughout growth (6,8). The vacuolar ATPase (vATPase), which pumps protons into the vacuole to maintain the vacuolar pH, has altered activity in btn1-{Delta} such that at elevated extracellular pH, vATPase activity is decreased, whereas proton pumping remains the same. This suggests that ATPase activity and proton transport coupling are disrupted when BTN1 is absent (8–11). Interestingly, plasma membrane ATPase activity and altered vacuolar pH in the early phases of growth in btn1-{Delta} can be reversed by artificially raising the vacuolar pH (12). Studies on CLN3 have also indicated that cells lacking CLN3 have increased lysosomal pH (13,14). Taken altogether, these observations indicate that CLN3 and Btn1p may be involved in lysosomal/vacuolar pH maintenance.

Btn1p has also been implicated in vacuolar arginine transport, as isolated vacuoles from btn1-{Delta} have a decreased rate of arginine uptake (5). However, Btn1p is unlikely to be a transporter, as Btn1p shares no sequence homology or motifs in common with any known transporter (15) and altered vacuolar arginine transport may result from the uncoupling of the vATPase activity and proton pumping (8). Nevertheless, lysosomes isolated from lymphoblast cell lines from Batten disease patients have decreased lysosomal arginine transport and decreased levels of intracellular arginine when compared with age- and sex-matched controls (16).

We have investigated how and why levels of arginine are decreased and whether there is a requirement to keep intracellular levels of this amino acid in check. Moreover, we have explored the important question as to whether restoration of arginine levels in this model would have some therapeutic value. To establish whether diminished intracellular arginine levels were due to altered flux of arginine in or out of the cell, we assayed the whole cell arginine uptake, efflux and incorporation into peptides and found no differences for btn1-{Delta} when compared with BTN1+. This suggested that arginine levels in btn1-{Delta} might be highly regulated and simply adding arginine to cells does not guarantee entry of this amino acid into the cell. We therefore overexpressed Can1p, the plasma membrane basic amino acid permease, to force the cells to increase their intracellular arginine. This resulted in a severe growth defect specific to btn1-{Delta} and to arginine. We have further uncovered that deletion of the master regulator of amino acid synthesis, GCN4 (17), in btn1-{Delta} results in a specific growth requirement for arginine and not other amino acids and that expression of BTN1 is both Gcn4p- and arginine-dependent. Taken together, these results show that arginine levels appear to be kept in check in btn1-{Delta} and the absence of Btn1p results in a precise requirement for regulation of intracellular basic amino acid levels. In addition, increasing intracellular levels of arginine suggest that supplementation of arginine in JNCL cells may be detrimental.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The yeast vacuole functions as a storage organelle for amino acids such as arginine and lysine (18–20). Similar to JNCL lymphoblasts, we have previously demonstrated that there is a defect in the transport of arginine into btn1-{Delta} vacuoles and a decrease in both cytosolic and vacuolar arginine levels (5,8).

What happens to the missing arginine?
It was unclear whether cytosolic arginine levels were lower in btn1-{Delta} as a result of altered transport into the vacuole. A comparison of gene expression between wild-type and btn1-{Delta} did not reveal any changes in genes involved in arginine synthesis or breakdown (6). We therefore investigated how these low levels are established. Plasma membrane arginine transport into the cell, which is primarily mediated by Can1p, could be decreased in btn1-{Delta}, thus accounting for the decreased cytosolic arginine levels if the change is mediated at the plasma membrane (21–23). Plasma membrane arginine transport was assayed in BTN1+ and btn1-{Delta} cells incubated with 135 µM arginine (Km = 10 µM) (22) for either 3 h (Fig. 1A and B) or 2 min (Fig. 1c). We had expected that perhaps btn1-{Delta} would exhibit a slower rate of transport than BTN1+, contributing to the decreased intracellular levels of arginine; however, there was no significant difference between BTN1+ and btn1-{Delta} arginine uptake, suggesting that plasma membrane arginine transport is normal in btn1-{Delta} (Fig. 1A–C).


Figure 1
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  		Figure 1. btn1-{Delta} does not have an altered rate of plasma membrane arginine uptake, efflux from the cell or arginine incorporation into peptides. Uptake—btn1-{Delta} (SP001) cell cultures (n = 6) were incubated with 135 µM 14C-arginine and unlabeled arginine. Measurements of radioactive uptake were normalized to buffer (no cell) control. Uptake was measured in can1-{Delta} (10305) as a negative control. (A) Curve representing plasma membrane arginine uptake from 0 to 180 min in nanomoles of arginine per cell. (B) Linear rate at which cells take up 135 µM arginine from surrounding media from 0 to 180 min in nanomoles of arginine per cell per minute. (C) Rates at which cells take up 135 µM arginine from surrounding media from 0 to 2 min in picomoles of arginine per cell per second. (D) Efflux—cold arginine was added to the uptake reaction (n = 4) at 60 min to measure efflux. Curve representing 14C efflux from 0 to 180 min in nanomoles of molecules per cell. (E) Linear rate at which the radiolabeled molecules are effluxed from the cell in nanomoles of molecules per cell per minute. (F) Incorporation—each strain was incubated with 14C-arginine (n = 3). Aliquots were TCA precipitated at 0, 30, 60 and 90 min. Linear rates of arginine incorporation are in nanomoles of arginine per cell per minute.

 
We then considered whether an increase in arginine efflux from the cells could account for decreased intracellular levels of arginine in btn1-{Delta}. Although very little is understood about amino acid efflux, an arginine efflux mechanism has been identified in yeast (24). We assayed arginine efflux by first incubating cells with radiolabeled arginine and subsequently adding unlabeled arginine to compete out the radiolabeled arginine. The radioactivity that remained in the cell was then measured. No significant difference was observed between BTN1+ and btn1-{Delta} for arginine efflux, suggesting that the decreased level of arginine in btn1-{Delta} does not result from increased efflux from the cell (Fig. 1D and E).

Finally, although a difference for only one amino acid would seem unlikely, we confirmed that there was no difference in protein incorporation of arginine in BTN1+ and btn1-{Delta} (Fig. 1f). A previous study revealed no differences in expression of genes involved in synthesis and utilization between BTN1+ and btn1-{Delta} (25); however, we conclude from these studies that a regulatory mechanism must exist, which works to keep intracellular arginine levels low in btn1-{Delta}.

btn1-{Delta} needs to be able to regulate arginine levels
To test the notion that there may be tight metabolic control of arginine levels in btn1-{Delta}, we deleted the master regulator of amino acid biosynthesis, GCN4, in a btn1-{Delta} strain (17). Gcn4p is a bZIP transcription factor that regulates the expression of many genes associated with amino acid synthesis, transport and storage in the response to starvation and stress conditions (26,27). An absence of Gcn4p causes an decrease in expression of genes involved in amino acid transport and synthesis. Therefore, we deleted BTN1 to uncover whether GCN4 had a role in regulating Btn1p. The btn1-{Delta} gcn4-{Delta} strain exhibited a growth defect specific to media lacking arginine (Fig. 2A and B). Furthermore, only the addition of L-arginine to the media restored growth of the btn1-{Delta} gcn4-{Delta} strain (Fig. 2c). Supplementation with precursors and products of arginine synthesis and catabolism, such as citrulline and ornithine as well as any other amino acids, did not rescue growth on media lacking arginine (Fig. 2C and data not shown). It should be noted that btn1-{Delta} cells are able to grow on media without arginine, indicating that decreased intracellular arginine levels are not due to a requirement for an extracellular source of arginine. Assessment of this growth in liquid media reveals that growth rates of wild-type and btn1-{Delta} cells are essentially identical (data not shown). It should also be noted that a gcn4-{Delta} mutant does display a slight growth defect on media lacking arginine, but that this growth defect is enhanced in btn1-{Delta} gcn4-{Delta}. To confirm that this growth defect was not an artifact of vacuolar dysfunction, we compared growth on media lacking arginine for a gcn4-{Delta} pep4-{Delta} and confirmed that deletion of PEP4 and GCN4 does not have the same growth defect as btn1-{Delta} gcn4-{Delta} (Fig. 2a). PEP4 encodes a protease required for vacuolar protease maturation, and deletion of this gene product results in a variety of vacuolar defects. In addition, deletion of VBA2, which encodes a known vacuolar arginine transporter (28), in combination with gcn4-{Delta}, vba2-{Delta} gcn4-{Delta}, did not result in a defect in growth on media lacking arginine (data not shown). We subsequently confirmed by comparative real-time reverse-transcriptase–PCR (RT2–PCR) a decrease in BTN1 mRNA levels in gcn4-{Delta}, indicating Gcn4p involvement in regulation of Btn1p levels. Furthermore, expression of BTN1 mRNA levels is elevated in the absence of arginine, although unaltered in response to elevated arginine (Table 1).


Figure 2
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  		Figure 2. btn1-{Delta} gcn4-{Delta} has a severe growth defect in the absence of arginine. A total of 107 cells were spotted in column 1, followed by sequential 10-fold dilutions in remaining columns. (A) btn1-{Delta} gcn4-{Delta} (SP003) is lethal on SC-arginine media. No arginine plates contain dextrose and all amino acids that are in the synthetic complete plates except arginine. Each row (left to right) shows decreasing amounts of cells (indicated by wedge). (B) Phenotype is specific to arginine. Strains were plated as described earlier onto SC media without phenylalanine, serine, tryptophans or lysine. (C) Cells were grown on SC-arg+L-arginine, lysine, D-arginine or citrulline. Only addition of L-arginine can rescue the growth defect.

 


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  		Table 1. BTN1 mRNA is increased in media lacking arginine and decreased in the absence of GCN4

 
Forced increase in intracellular arginine is toxic to btn1-{Delta}
We have previously demonstrated that lymphoblasts derived from JNCL patient cells have decreased lysosomal arginine transport and an overall decrease in cellular levels of arginine (16). A key question is whether this decreased level of arginine in JNCL cells is a potential contributor to the disease and whether efforts should be made to raise intracellular levels of arginine as a means to treat the underlying defect associated to JNCL. A second consideration that is equally as important is whether intracellular levels of arginine are kept low to compensate for the defect in CLN3 and thus act in a somewhat protective manner in JNCL.

Having established the precedent that arginine levels appear to have the potential for tighter than normal regulation in btn1-{Delta}, we have exploited decreased arginine levels in btn1-{Delta} as a means to establish whether decreased arginine levels in JNCL should be targeted for correction. However, we observed no difference in the growth of btn1-{Delta} when compared with BTN1+ in the presence of high arginine levels, up to 65 mM arginine, with arginine becoming lethal between 2.5 and 25 mM for both BTN1+ and btn1-{Delta} (Fig. 3a). Exposure to 50 mM arginine in liquid culture for 4 h followed by plating onto normal media does not reveal any differences in BTN1+ and btn1-{Delta} viability (Fig. 3b). Note btn1-{Delta} does not require arginine for growth (Fig. 3c).


Figure 3
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  		Figure 3. btn1-{Delta} and wild-type cells are equally sensitive to elevated levels of arginine, and btn1-{Delta} does not have a growth requirement for arginine. (A) Serial dilutions of BTN1+ (B11718) and btn1-{Delta} (SP001) cells were plated on 0.1, 1, 2.5, 25 and 65 mM arginine plates. (B) BTN1+ (B11718) and btn1-{Delta} (SP001) cells were incubated in SC-arginine with 50 mM arginine added back and plated at 4 h. Colonies were counted and normalized to 0 h. (C) Serial dilutions (indicated by wedge) of BTN1+ (B11718) and btn1-{Delta} (SP001) cells grown in the absence of arginine.

 
We next considered that our test of whether growth was affected on high arginine media was incomplete, as it did not necessarily result in an elevation of intracellular arginine. Therefore, in order to force the cell to take up more arginine, we overexpressed CAN1, which encodes the plasma membrane basic amino acid transporter, responsible for 96% of cellular arginine uptake, in btn1-{Delta} and BTN1+ cells (21–23). Thus, we could directly test whether higher intracellular arginine levels are toxic to btn1-{Delta} cells. Interestingly, overexpression of CAN1 caused a dramatic and significant (P < 0.001) growth defect in btn1-{Delta}, but not in BTN1+ cells (Fig. 4A and B). To confirm that overexpression of Can1p does result in increased arginine uptake, we measured arginine uptake in BTN1+ cells overexpressing CAN1 and surprisingly found no difference from the vector control (Fig. 4c). Attempts to measure uptake in btn1-{Delta} were unsuccessful. After only a 2 h induction of CAN1, the results were confounded because of obvious cell death exhibited by this strain. In addition, closer examination of the btn1-{Delta} cells revealed a significant increase in the size of btn1-{Delta} cells compared with BTN1+ upon overexpression of Can1p (Fig. 5A and B). Moreover, use of the weaker MET promoter gave similar confounding results. Note in all case these cell population did not grow.


Figure 4
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  		Figure 4. CAN1 overexpression results in a severe growth defect specific for btn1-{Delta}. Cells were grown at 30°C in media containing galactose (SCG-ura) unless otherwise indicated. Readings at OD600 were taken over 42 h (A) Growth curve over 42 h. Squares are BTN1+ with vector (DW001), triangles are BTN1+ overexpressing CAN1 (DW002), inverted triangles are btn1-{Delta} with vector (DW004) and diamonds are btn1-{Delta} overexpressing CAN1 (DW005). (B) Growth rates calculated from log growth phase from (A). (C) Rates of arginine uptake in BTN1+ with vector or overexpressing CAN1. Cell cultures (n = 4) were incubated with 10 µM 14C-arginine and uptake was performed as previously described.

 


Figure 5
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  		Figure 5. CAN1 overexpression increases cell size in btn1-{Delta}. (A) Mean size of BTN1+ or btn1-{Delta} cells overexpressing CAN1 (n = 10). (B) Photomicrographs of BTN1+ and btn1-{Delta} cells upon overexpression of CAN1. Both top and bottom panels show overexpression of CAN1.

 
Btn1p is a vacuolar protein (6,7). To confirm that the growth defect for btn1-{Delta} overexpressing Can1p was specific to btn1-{Delta} and not a result of altered vacuolar function, we overexpressed Can1p in other vacuolar mutants. CUP5 (VMA3) encodes subunit c of the vATPase V0 subunit, VBA2 encodes a putative vacuolar arginine permease and PEP4 (PRA1) encodes a protease required for vacuolar protease maturation (28–34). Overexpression of CAN1 in the vacuolar mutants cup5-{Delta}, vba2-{Delta} and pep4-{Delta} did not result in the same growth defect, indicating that the btn1-{Delta} defect was not due to overall vacuolar dysfunction, but instead was specific to btn1-{Delta} (Fig. 6a). To confirm that the CAN1 overexpression growth defect was specific to CAN1, we also overexpressed IST2, another integral plasma membrane protein which is a putative ion channel (35). A minor growth defect was observed upon overexpression of IST2; however, the defect was not nearly as severe as the CAN1-dependent defect and not significantly different between BTN1+ and btn1-{Delta} (data not shown). Thus, the growth defect we observe is not an artifact of vacuolar dysfunction, rather it is specific to lacking Btn1p.


Figure 6
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  		Figure 6. Lethality upon CAN1 overexpression is not due to a vacuolar defect or the presence of arginine alone. (A) Growth rate for vacuolar mutants cup5-{Delta} (10268), pep4-{Delta} (12098), vba2-{Delta} (15741) with vector or CAN1 overexpression (DW006–011). (B) Growth rate of btn1-{Delta} overexpressing CAN1 in the presence or absence of arginine.

 
Can1p transports all basic amino acids, not just arginine (36). To verify that defective growth for btn1-{Delta} was specifically due to increased arginine transport upon overexpression of CAN1, we repeated the experiment in media lacking arginine. Unexpectedly, the growth defect was not rescued by removing arginine from the media (Fig. 6b). The fact that the removal of arginine from the media did not restore growth for btn1-{Delta} overexpressing Can1p was puzzling, but suggested that one or a combination of the other substrates for Can1p, namely lysine or histidine, could result in the severe growth defect for btn1-{Delta} overexpressing CAN1. The strain used for this experiment is his3-{Delta} and lys2-{Delta} and therefore has a growth requirement for histidine and lysine. Thus, we could not remove histidine or lysine from the media to test whether uptake of these amino acids affects the growth defect. Owing to the metabolic requirements of this strain background (Table 2), we utilized a btn1-{Delta}::HIS3, LYS2 (B-10195) strain in order to remove all Can1p substrates (arginine, lysine and histidine) from the media. We tested the growth of B-10195 overexpressing Can1p in the presence of arginine, lysine and histidine compared with an empty vector and demonstrated specifically that arginine and lysine are toxic to this strain (Fig. 7). Interestingly, the presence of histidine, a competitive inhibitor for arginine and lysine transport (22), rescued growth in the presence of either arginine or lysine, further indicating that arginine or lysine can indeed cause the growth defect for btn1-{Delta}. It should be noted that in the previous figure, histidine did compete out toxicity of arginine in the histidine auxotroph, indicating that substituting amino acids in media is not the same as having an intact biosynthetic pathway. In addition, under these conditions, the high-affinity histidine transporter, Hip1p, is the likely uptake mechanism for this amino acid. Thus, in the absence of auxotrophic requirements that typically complicate studies on laboratory strains of yeast, we conclude that elevating intracellular levels of arginine and/or lysine in btn1-{Delta} are toxic to the cell.


Figure 7
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  		Figure 7. CAN1 overexpression results in a severe growth defect specific for btn1-{Delta}, which is dependent on arginine or lysine. BTN1+ and btn1-{Delta} cells containing either vector or CAN1 construct (DW012–015) were grown in media synthetic complete containing galactose without uracil (SCG-ura), with lys, his and arg added where indicated at 30°C. OD600 measurements were taken over 48 h. (A) Growth curve of cells in SCG-ura-his-lys media (of the three basic amino acids, only contains arginine). (B) Growth curve of cells in SC-ura-his-arg (only lysine added). (C) Growth curve of cells in SCG-ura-lys-arg (only histidine added). (D) Growth curve of cells in SCG-ura-lys (histidine and arginine added). (E) Growth curve of cells in SCG-ura-arg (histidine and lysine added). (F) Growth curve of cells in SCG-ura-his (lysine and arginine added). Growth curves representative of three independent trials.

 


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  		Table 2. Yeast strains used in this study

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Vacuoles of yeast lacking Btn1p have a decreased pH at the early log phase and this low vacuolar pH increases throughout growth to elevated levels (6). Moreover, isolated vacuoles from btn1-{Delta} have a decreased rate of arginine uptake, and there is an overall decrease in levels of vacuolar and, to a lesser extent, cytosolic arginine when compared with BTN1+ (5). Collectively, these observations led to three questions. First, why are arginine levels decreased in btn1-{Delta} and what happens to the missing arginine? Secondly, what happens if we elevate the cellular levels of arginine in btn1-{Delta}? Finally, how do we apply this to CLN3 and JNCL?

With regard to why arginine levels are low in btn1-{Delta}, we have shown that this does not result from decreased uptake into the cell, increased efflux from the cell or elevated incorporation into peptides. Therefore, decreased intracellular arginine levels in btn1-{Delta} likely arise from decreased synthesis or increased utilization of arginine, independent of protein synthesis and level of Can1p which transports arginine into the cell. This suggests that the absence of Btn1p predicates a requirement for keeping intracellular levels of arginine and lysine levels down and that sufficient levels of arginine uptake are maintained by the previously reported increase in BTN2 mRNA levels in btn1-{Delta} (Fig. 8) (5,6). Btn2p was shown to interact with Rhb1p, a small GTPase tethered to the membrane through farnesylation, which, among other functions, is implicated in the upstream negative regulation of Can1p activity (37,38). We previously reported that btn2-{Delta} and rhb1-{Delta} single and double mutants exhibited increased sensitivity to canavanine, a toxic arginine analog, suggesting that Btn2p participates in the regulation of Can1p, most likely through Rhb1p. How this regulation occurs is still unknown.


Figure 8
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  		Figure 8. Mechanism of arginine and lysine toxicity in btn1-{Delta}. Overexpression of CAN1 is lethal in btn1-{Delta}. There are three mechanisms for controlling excess Can1p in cells that may contribute to arginine toxicity: regulating the amount of Can1p at the plasma membrane, regulating the Can1p activity itself and storing excess arginine in the vacuole. (A) (Wild-type) In a normal cell, excess Can1p can be sequestered in the Golgi (1). If excess Can1p does reach the plasma membrane, BTN1+ cells have a functional Btn1p that allows normal sequestration of arginine into the vacuole (2) and normal levels of Btn2p and free Rhb1p that allow regulation of Can1p activity (3). (B) (btn1-{Delta}) In btn1-{Delta}, Can1p may be sequestered in the Golgi (4); however, increased levels of Btn2p sequester increased amounts of Rhb1p, resulting in a loss of regulation of Can1p activity (5) causing elevated cytosolic levels of arginine and lysine that become toxic to the cell (6). The absence of Btn1p decreases the ability to sequester excess arginine in the vacuole (7). (C) (btn1-{Delta}) Alternatively, Can1p is not retained at the Golgi (8) and is transported to the plasma membrane (9). Excess Can1p at the plasma membrane intensifies the lack of Can1p regulation by Rhb1p and therefore arginine uptake into the cell (10) as well as the lack of arginine sequestration in the vacuole (11).

 
We postulated that there must be a compensatory mechanism keeping arginine levels at a tolerable level in btn1-{Delta} and that there was a threshold of arginine and lysine concentrations that would cause toxicity. It has been previously reported that overexpression of CAN1 in wild-type cells results in Can1p being sequestered in the Golgi such that the amount of Can1p at the plasma membrane is kept in check (39). We found further evidence that this is likely, as there is no increase in Can1p activity upon overexpression in BTN1+. We conclude that if we overexpress CAN1 in btn1-{Delta}, btn1-{Delta} cannot control either the amount of Can1p at the membrane or the Can1p activity (Fig. 8). A plausible explanation of the latter hypothesis would be that it has previously been shown that BTN2 is upregulated in btn1-{Delta} (6) and that Btn2p interacts with and is required for Golgi localization of Yif1p, which interacts with Yip1p (40,41). Yip1p, in turn, is required for recruitment of transport GTPases and proper ER to Golgi transport (42). Therefore, alterations in transport GTPases in btn1-{Delta} may precipitate more Can1p released from the Golgi to the plasma membrane. Thus, btn1-{Delta} cells take up too much arginine and lysine, swell and die. Swelling in btn1-{Delta} could result from ionic imbalances due to increased cytoplasmic cationic content or alkalinity. We therefore attempted to localize overexpressed functional GFP-tagged Can1p upon induction in btn1-{Delta}, but this proved futile as the cells were dead and a definitive localization using microscopy or western blot for the GFP tag was inconclusive. It should be noted that endocytosis, vesicle fusion and protein degradation at the vacuole could also be defective under these conditions.

Comparison of gene expression between btn1-{Delta} and BTN1+ cells did not reveal an altered expression of genes involved in arginine/lysine or any other amino acid synthesis or utilization (25). Therefore, the alteration in arginine/lysine levels is not likely brought about at the transcriptional level. Natarajan et al. (27) have implicated Gcn4p in a plethora of pathways along with amino acid regulation redundant pathways, so we cannot rule out another Gcn4p-dependent pathway contributing to our observation. However, it is compelling that btn1-{Delta} gcn4-{Delta} strains fail to grow in the absence of arginine. It was previously shown that elevated uptake of arginine in a btn2-{Delta} rhb1-{Delta} strain that loses the ability to negatively regulate arginine uptake through Can1p is suppressed by btn1-{Delta}, strongly suggesting that the absence of Btn1p does predicate a requirement for keeping intracellular levels of arginine and lysine levels down (38). Deletion of GCN4 has been reported to cause accumulation of vacuolar amino acids under starvation conditions. Gcn4p is central to the control of amino acid biosynthesis (43), and although it is not clear why gcn4-{Delta} accumulate amino acids in the vacuole (44), it is apparent that this accumulation is at odds with the decrease in vacuolar amino acids which results from btn1-{Delta}. gcn4-{Delta} would certainly result in altered regulation of amino acid anabolism and catabolism. Therefore, if btn1-{Delta} strains have a requirement for keeping the level of amino acids such as arginine low in the vacuole, the loss of control of amino acid synthesis/degradation by the introduction of gcn4-{Delta} results in an inability to grow. This is supported by the slight growth defect we see for gcn4-{Delta} on media lacking arginine. The elevated vacuolar arginine in gcn4-{Delta} may act as a reserve in the absence of arginine. In contrast, a btn1-{Delta} strain has a decrease in vacuolar arginine levels. A gcn4-{Delta} strain can grow better than the btn1-{Delta} gcn4-{Delta} double deletion because the gcn4-{Delta} cells can utilize the vacuolar arginine reserve and thus survive a few generations, albeit slowly, in the absence of extracellular arginine. For btn1-{Delta} gcn4-{Delta}, there is no stored arginine and therefore the cells cannot import it, cannot synthesize it and cannot use any arginine stored in the vacuole.

BTN1 expression in gcn4-{Delta} is decreased, suggesting that at some level BTN1 is a regulatory target of Gcn4p. BTN1 has five putative Gcn4p binding sites upstream of the ORF as determined by in silico analysis, although this same analysis also revealed several other transcription factor sites (data not shown). However, further studies on the BTN1 promoter region will likely be required to verify whether BTN1 is a true target for Gcn4p.

The first question we asked was why are arginine levels decreased in btn1-{Delta} and where does the normal level of arginine go? It would seem that the answer is quite complex, as arginine levels are tightly regulated and it is apparent that this regulation may have to be tighter in btn1-{Delta}. A simpler interpretation is that increased arginine levels are toxic in btn1-{Delta} because the strain is not able to compensate for increases. Metabolic profiling of btn1-{Delta} compared with normal cells may reveal that activities that rely on arginine utilization, such as synthesis of nitric oxide and polyamines for example, result in a shift in other metabolites.

A key unresolved question is why do normal strains sequester arginine and lysine in the vacuole? The R groups of these amino acids have additional nitrogen which may imply that accumulation of these amino acids might be for nitrogen storage. Certainly, GCN4 has been broadly implicated in metabolic regulation. However, previous studies have not revealed that btn1-{Delta} has a growth phenotype when limited for nitrogen. We propose that sequestration of arginine in the vacuole may also have something to do with cationic balance in the cell, rather than or as well as a means to store nitrogen. We show that arginine availability clearly directs BTN1 expression as there is an upregulation of BTN1 in cells grown in the absence of arginine. In addition, we have answered our second question which was what happens if we elevate the cellular levels of arginine in btn1-{Delta}? We observed that overexpression of CAN1 and thus increased uptake of arginine or lysine is lethal in btn1-{Delta}. The fact that this growth defect was not evident when we examined increased arginine uptake for other vacuolar mutants strengthens the role that Btn1p might play in regulating intracellular arginine levels. We had previously suggested that altered arginine levels in btn1-{Delta} might result in an ionic imbalance of the vacuole, as these are the main sequestered amino acids (45). The basis of this assertion is that as well as being incorporated into proteins and serving as a precursor to other biochemical pathways, in this case, arginine could simply be acting as an organic cation. btn1-{Delta} has also been shown to have altered vacuolar pH. Therefore, future experiments will focus on exploring a possible role for Btn1p at the vacuole in regulating the cationic strength of vacuolar lumenal contents.

So what does this mean for CLN3 and JNCL? Yeast and humans bear many metabolic differences, and importantly, arginine is an essential amino acid for humans, but not for yeast. Cellular response to altered arginine between yeast and humans differs in the regulation of the plasma membrane group the arginine permeases, Can1p in yeast, and the CAT transporters which transport arginine in human cells (21–23). The amount of Can1p at the plasma membrane is kept in check by retention in the Golgi, and the activity is regulated by Rhb1p (37,39). Although very little is known about how CAT transporters are regulated, CAT-1 is thought to be regulated by gene expression, mRNA turnover and translation (46,47). Although similar to the AP-1 transcription factors and a member of the basic leucine zipper family, there is no known mammalian homolog to Gcn4p, and examination of CLN3's upstream theoretical promoter region reveals no Gcn4p binding sites (26,48). Thus, a more in-depth analysis of the CLN3 promoter region is needed. Although it is clear that Btn1p and CLN3 have functional conservation and that differences exist for both btn1-{Delta} and JNCL cells with regard to intracellular levels of arginine, how yeast and human cells accommodate altered arginine likely differs. Despite these differences, our findings indicate that arginine levels in btn1-{Delta} require precise regulation, that manipulation of intracellular arginine levels may be detrimental to cell viability and that manipulation of arginine levels in JNCL warrants caution.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast strains, media and plasmid construction
The B11718 [GenBank] wild-type strain was purchased from RESGEN. The KANMX system was used to delete BTN1 using homologous recombination, followed by Cre-recombinase-driven excision of the KANr cassette, as described by Guldener et al. (49). Double deletions were made in a similar manner after the kanamycin resistance cassette was excised at the first allele. Single and double deletions were confirmed by PCR. All other single mutant strains were purchased from the deletion consortium (RESGEN or ATCC).

Media and growth conditions
Standard yeast media was used in all growth experiments unless otherwise specified. Synthetic complete media contained 6.7 mg/ml yeast nitrogen base without amino acids, 5 mg/ml ammonium sulfate, 2% dextrose and all amino acids except asparagine, glutamine, proline, alanine, cystine and glycine. Plasmids were maintained using a uracil auxotrophic marker in synthetic complete media lacking uracil (SC-ura). Arginine omission plates were made using synthetic complete mix without arginine. Induction media for overexpression plasmids contained 2% galactose, 0.1% raffinoses and synthetic complete mix without the indicated amino acids. Minimal induction media contained 6.7 mg/ml yeast nitrogen base with amino acids, 5 mg/ml ammonium sulfate, 2% galactose, 0.1% raffinose, 30 mg/l leucine and 20 mg/l tryptophan plus the indicated amino acids. The pH of all media (except YPD) was adjusted to 6 before use. For plate phenotypes, strains were in grown in YPD overnight, harvested and washed twice with sterile water. Cells were resuspended to 3 x 108 cells/ml and diluted 10-fold for each column. A 36-pin replicator was used to spot cells onto the media, followed by incubation at 30°C for 5 days. To construct growth curves, strains were transformed with plasmids using the standard lithium acetate technique (50,51). The resulting strains were grown in SC-ura to OD600 0.6–1.0, harvested, washed twice with sterile water and resuspended in the indicated induction media to OD600 0.1. Cultures were incubated in a 30°C rotor and growth was recorded by measuring the OD600 over a 48 h period. Rates of growth were determined by calculating the slope during log phase. Significance was determined by Student's t-test (P ≤ 0.05).

Plasmid construction
Plasmids DAP229 and DAP182 were constructed by PCR amplification of CAN1 from yeast chromosomal DNA and subcloned into pCR-Zero blunt (Invitrogen), excised and ligated into the GAL inducible vector pAA1052/pYeura3 (Clontech) using the Xba1/Xho1 sites. This construct complemented for a CAN1+-dependent growth defect on media containing canavanine, a toxic analog of arginine (data not shown) which shows that our construct encodes a functional Can1p.

Cell diameter measurements
Epifluorescent microscopy was used to take differential contrast images. Cells were chosen at random and measured using ImageJ software (NIH). Significance was determined by Student's t-test (P = 0.0014).

Arginine transport, efflux and incorporation
For uptake, cells were grown to OD600 0.6, harvested and resuspended to OD600 2.0. Arginine transport at the plasma membrane was measured using techniques adapted from Urano et al. (37) and Chattopadhyay and Pearce (38). Each experiment was repeated independently three to six times to determine statistical significance. Cells were aliquoted to 1.4 ml and 200 µl was pipetted onto a Whatman GF/C filter on a Millipore vacuum manifold for the 0 s/min time point. To each sample, 15 µl of L-[U-14C] arginine (300 mCi/mmol, Amersham Biosciences) and 12 µl 10 mM unlabeled L-arginine (135 µM total substrate) was added to start the reaction. Aliquots were taken as described earlier at various time points. Reactions were stopped by washing twice with cold synthetic media (2% dextrose, 20 mg/ml histidine, 20 mg/ml uracil, 20 mg/ml tryptophan). Radioactivity was measured via scintillation. GraphPad Prism® was used for graphing and statistical analysis. A Student's t-test was performed and a difference was deemed significant if P < 0.05.

Efflux was measured as described by Opekarova and Kubin (24) with some adaptations. Cells were grown, harvested and aliquoted as in arginine uptake. Cells were incubated with arginine as described earlier for 60 min when group cold arginine 50 mM. Aliquots, measurements and data analysis were performed as described earlier.

Arginine incorporation was measured by TCA precipitation. Cells were grown to OD600 0.6 or 6.0, harvested, incubated in SC-arg media for 40 min at 30ºC and aliquoted into 1 ml samples. Aliquots of 5 µl of L-[U-14C] arginine (300 mCi/mmol, Amersham Biosciences) and 10 µl of 10 mM unlabeled L-arginine were added to start the reaction. Aliquots were incubated at 30°C and quick frozen on dry ice at 0, 30, 60 and 180 min. At the end of the reaction, 5 ml 5%TCA was added. The samples were heated at 90°C for 20 min, followed by a 30 min incubation on ice. Precipitate was collected on Whatman GF/C filters using a Millipore vacuum manifold, followed by washing with 20 ml of 95% ethanol. Radioactivity was measured via scintillation, and rates were calculated as described earlier.

Quantification of BTN1 mRNA levels
RNA was extracted from GCN4+, gcn4-{Delta} or btn1-{Delta} yeast using a phenol, glass bead protocol. Forty units of yeast cells were bead beaten in the presence of 25:24:1 phenol:chloroform:isoamyl alcohol equilibrated with RNA buffer (0.2 M Tris–HCl, pH 7.5, 0.5 M NaCl, 0.01 M EDTA, 0.1% SDS) and ß-mercaptoethanol, ethanol precipitated and quantitated using Quant-iTTM RiboGreen RNA assay kit as per manufacturer's protocol (Molecular Probes) and treated with DNaseI (Fermentas). Random hexamer priming with or without reverse transcriptase was used to synthesize cDNA from 5 µg RNA using the First strand Synthesis for rt-PCR kit according to the manufacturer's protocol (Invitrogen). Transcript levels were measured using comparative RT2–PCR. RT2–PCR compares the amount of a specific transcript, such as BTN1, in two different conditions. The transcript is normalized to a loading control such as ACT1 (actin), the final aim being to compare gene regulation in different conditions such as wild-type versus mutant or normal versus altered growth conditions. Reactions containing 2 µl cDNA, 0.25 µM primers specific to ACT1 (Forward: atg gtc ggt atg ggt caa aa; Reverse: aac cag cgt aaa ttg gaa cg) or BTN1 (Forward: cct gac tta cca aag tct tc; Reverse: tca ttc tca taa gat gtc ca) and iQTM SYBR® Green Supermix (BioRad) were run on an icycler (BioRad) using the following reaction parameters: 95°C, 2 min, 1 cycle, then 95°C for 20 s, 51°C for 20 s, 72°C for 45 s, 40 cycles followed by 95°C for 1 min and 55°C for 1 min. Changes in transcript levels were analyzed using REST© software (52). PCR efficiencies were analyzed using DART-PCR© software (53), with both sets of primers having 100% efficiency.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Danyra Chavez for her assistance in the growth curve experiments, Jared Benedict for help in creating the figures and Sergio Padilla-Lopez for his helpful comments. Supported by NIH grant R01 NS36610.

Conflict of Interest statement. None declared.


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