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Vol. 17, Issue 10, 4411-4419, October 2006
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Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Submitted June 8, 2006;
Revised July 19, 2006;
Accepted July 26, 2006
Monitoring Editor: David Drubin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The activity of many amino acid permeases is regulated by the quantity of available amino acids as well as the quality of the nitrogen source. Genes encoding several amino acid permeases, such as AGP1, GNP1, BAP2, BAP3, DIP5, TAT1, and TAT2, are transcriptionally induced by the presence of extracellular amino acids (Didion et al., 1998; Iraqui et al., 1999). This transcriptional induction requires the extracellular SPS amino acid sensor that proteolytically activates the transcription factor Stp1p in the presence of amino acids (Forsberg and Ljungdahl, 2001; Andreasson and Ljungdahl, 2002). There is also evidence that the Tat2p and Bap2p permeases are regulated post-translationally such that these permeases are only present at the plasma membrane when their substrate amino acids are available for import from the extracellular medium (Beck et al., 1999; Omura et al., 2001).
The subset of permeases induced by elevated amino acid levels include both specific and general amino acid transporters, but all have a relatively low capacity for transport. In contrast, the activity of the general amino acid permease (GAP1), which is responsible for the high-capacity transport of all naturally occurring amino acids for use as a nitrogen source, is repressed by amino acids both transcriptionally and post-transcriptionally through a sorting process in the late secretory pathway (Grenson et al., 1970; Chen and Kaiser, 2002).
GAP1 is transcribed by two GATA transcription factors: Gln3p, which is repressed in glutamine or ammonia medium, and Nil1p, which is repressed by elevated levels of glutamate or any other amino acid (Stanbrough et al., 1995; Magasanik and Kaiser, 2002; Risinger and Kaiser, unpublished data). Therefore, the GAP1 gene product is produced when the nitrogen source in the growth medium is poor or when total intracellular amino acid levels are low.
Gap1p is an integral membrane protein that is transported through the secretory pathway to the trans-Golgi. At the trans-Golgi, Gap1p is either delivered to the plasma membrane, where it can import amino acids from the medium, or sent to the vacuole for degradation. Poly-ubiquitination of Gap1p by the Rsp5p-Bul1p-Bul2p ubiquitin ligase complex at the trans-Golgi is required for targeting of the permease to the prevacuolar endosome; mutation of the ubiquitin ligase machinery (bul1
bul2) or the ubiquitinated lysine residues of Gap1p (GAP1K9R,K16R) results in constitutive plasma membrane localization of Gap1p (Helliwell et al., 2001; Soetens et al., 2001). When Gap1p is ubiquitinated and sorted to the prevacuolar endosome, it can recycle back to the trans-Golgi for another attempt to reach the plasma membrane; it is this recycling step that is blocked by the presence of amino acids (Chen and Kaiser, 2002; Rubio-Texeira and Kaiser, 2006). Therefore, elevated internal amino acid levels cause any expressed Gap1p to be sorted to the vacuole, resulting in low amino acid transport through Gap1p (Stanbrough and Magasanik, 1995; Chen and Kaiser, 2002). However, when internal amino acid levels are scarce, Gap1p is able to reach the plasma membrane where it can scavenge any available amino acids in the medium through high-affinity transport.
We were interested in finding a physiological rationale for Gap1p repression in response to elevated internal amino acid levels given that the activity of most other amino acid permeases is induced by amino acids. Surprisingly, when we explored the physiological consequences of disrupting Gap1p regulation in response to amino acids, we discovered a novel mechanism of amino acid–dependent repression of Gap1p activity: rapid and reversible inactivation of amino acid transport through the permease at the plasma membrane. Interestingly, we also found that exposure of cells expressing Gap1pK9R,K16R to any one of a number of individual amino acids caused a rapid cessation of growth and a loss of viability despite the ability of the permease to be inactivated at the plasma membrane.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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) in pRS316. The plasmid was cleaved in the 5' sequence with EagI and within the GAP1 ORF with Bsu36I and transformed into either wild-type or sec6-4 strains, where the fragment homologously recombined with the endogenous GAP1 allele, replacing it with the new version and inserting the kanMX6 gene upstream of the promoter. Lysine mutants were constructed in a similar manner by performing Stratagene Quick-Change site-directed mutagenesis (La Jolla, CA) on the above plasmids to introduce the lysine mutations before homologous recombination. Plasmids used in this study were pAR70, GAP1 under its own promoter in a URA3-CEN vector; pAR73, GAP1K9R,K16R under its own promoter in a URA3-CEN vector; pEC221, ADH1 promoted GAP1 in a URA3-CEN vector; pAR1, ADH1 promoted GAP1K9R,K16R-HA in a URA3-CEN vector; pAR13, ADH1 promoted GAP1-GFP in a URA3-CEN vector; pAR14, ADH1 promoted GAP1K9R,K16R-GFP in a URA3-CEN vector; pNC3, ADH1 promoted gap1V363G in a URA3-CEN vector; pNC4, ADH1 promoted gap1L185V in a URA3-CEN vector; pNC5, ADH1 promoted gap1A497V in a URA3-CEN vector; pNC6, ADH1 promoted gap1A365V,T590A in a URA3-CEN vector; pNC7, ADH1 promoted gap1A297V in a URA3-CEN vector; and pNC8, ADH1 promoted Gap1K9R,K16R,A297V-HA in a URA3-CEN vector.
Minimal (SD) medium is composed of Difco yeast nitrogen base without amino acids and without ammonium sulfate, 2% glucose, 0.5% ammonium sulfate (adjusted to pH 4.0 with HCl; Difco, Detroit, MI). Individual amino acid stocks were made at 40–200 mM in SD medium at pH 4.0, filter-sterilized, and stored at 4°C. Casamino acid medium contains SD with Casamino acids (Difco) added from a 10% stock (pH 4.0) to a final concentration of 0.25 or 0.0025%.
Screen for Gap1p Transport Mutants
GAP1 mutations were generated by mutagenic PCR using pEC221 (PADH1-GAP1) as a template and methods described previously (Sevier and Kaiser, 2006) with modifications. A fragment including the entire GAP1 ORF as well as 500 base pairs of the ADH1 promoter and 800 base pairs of the GAP1 3' UTR was amplified in four 50-µl reactions with AmpliTaq Gold (Perkin-Elmer Cetus, Norwalk, CT) and 0.1 mM MnCl2. PCR products were transformed along with gapped pEC221 plasmid (lacking the GAP1 ORF) into CKY701 (bul1 bul2 gap1 ura3-52), and gap-repaired plasmids were isolated by selection for Ura+ transformants. Citrulline-resistant transformants were identified by replica plating onto SD with 4 mM citrulline at 30°C. Resistant clones were then tested for sensitivity to glycine by replica plating to SD with 1 mM glycine. Plasmids were isolated from citrulline-resistant, glycine-sensitive colonies, retransformed into CKY701, and retested for citrulline and glycine sensitivity. Plasmids conferring resistance to citrulline and sensitivity to glycine arose at a frequency of 
10–3.
Amino Acid Uptake Assays
Strains were cultured to 4–8 x 106 cells/ml, subjected to indicated treatment, and washed with nitrogen-free medium by filtration on a 0.45-µm nitrocellulose filter before amino acid uptake assays were performed as described previously (Roberg et al., 1997). The specific activity of glycine was 112 mCi/mmol.
Amino Acid Accumulation Assays
GAP1K9R,K16R (CKY893) was cultured at a concentration of 5 x 106 cells/ml in minimal SD medium and distributed into 1-ml aliquots. [14C]glycine, [14C]-lysine, [14C]-threonine, or [14C]lysine were added either alone or in combination with the other three unlabeled amino acids to a final concentration of 1 mM. The total accumulated radiolabeled amino acid was measured after 20 min.
Fluorescence Microscopy
Strains expressing PADH1-GAP1-GFP or PADH1-GAP1K9R,K16R-GFP were cultured overnight in minimal SD medium to exponential phase at 24°C. Cells were harvested, resuspended in 300 mM Tris, pH 8, with 1.5% NaN3, and visualized using a fluorescence microscope. Images were captured with a Nikon E800 microscope (Melville, NY) equipped with a Hamamatsu digital camera (Bridgewater, NJ). Image analysis was performed using Improvision OpenLabs 2.0 software (Lexington, MA).
Equilibrium Density Centrifugation and Antibodies
Yeast membranes were fractionated by equilibrium density centrifugation on continuous 20–60% sucrose gradients containing EDTA as described (Kaiser et al., 2002). Antibodies used were as follows: rabbit anti-Gap1p; rabbit anti-Pma1p (gift of S. Losko and R. Kolling, Dusseldorf, Germany); and horseradish peroxidase–coupled sheep anti-rabbit (Amersham Pharmacia, Piscataway, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chen and Kaiser, 2002). As a control to show that this mutant no longer responded to high intracellular levels of amino acids, we found Gap1p-dependent citrulline uptake activity of PADH1-GAP1K9R,K16R remained high in an mks1 strain (Figure 1). MKS1 is a negative regulator of the Rtg1/3 transcription factors that are responsible for synthesis of 
-ketoglutarate, an amino acid precursor (Dilova et al., 2002; Sekito et al., 2002) and mks1 strains have elevated internal amino acid concentrations sufficient to cause Gap1p to be sorted to the vacuole in a wild-type cell (Figure 1; Chen and Kaiser, 2002; Rubio-Texeira and Kaiser, 2006). The finding that the localization and activity of Gap1pK9R,K16R was not perturbed by elevated internal amino acid levels supports the hypothesis that Gap1p must first be ubiquitinated before sorting can be regulated by amino acids levels.
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mutation, when a rich mixture of amino acids (0.25% Casamino acids) was added exogenously to a strain expressing PADH1-GAP1K9R,K16R, amino acid import through Gap1pK9R,K16R was very low (Figure 1). We found Gap1pK9R,K16R localized to the plasma membrane both by fractionation and fluorescence microscopy in the presence of Casamino acids (Figure 2, A and B), suggesting that exogenously added amino acids could inactivate amino acid import through Gap1p that resided at the plasma membrane. To explore this possibility further, we followed the change in Gap1pK9R,K16R activity with time after the addition of 0.25% Casamino acids to the medium. Immediately after amino acid addition, amino acid import through Gap1pK9R,K16R remained high, indicating that exogenously added Casamino acids were not simply blocking [14C]citrulline uptake by competitive inhibition (Figure 3A). The rate of [14C]citrulline uptake through Gap1pK9R,K16R decreased with time, such that 1 h after amino acid addition Gap1p activity had decreased to <10% the starting activity (Figure 3A). We conclude that the presence of extracellular amino acids was sufficient to inactivate amino acid transport through plasma membrane localized Gap1p, uncovering a new and distinct mechanism of amino acid–dependent down-regulation of Gap1p activity.
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Amino Acid–dependent Inactivation of Gap1p Is Reversible
To determine whether the inactivation of Gap1p was reversible, we utilized a temperature sensitive sec6-4 strain that blocks delivery of newly synthesized protein to the plasma membrane at its restrictive temperature (Novick et al., 1980; Walworth and Novick, 1987). When amino acids were added to sec6-4 strains expressing wild-type Gap1p, we observed a rapid decrease in Gap1p activity (Figure 4A) that corresponded to a loss of the permease from the plasma membrane due to ubiquitin-mediated endocytosis (Figure 4B). Twenty minutes after amino acid addition (a time when most of Gap1p had been removed from the plasma membrane), cells were shifted to 36°C for 10 min, washed, and resuspended in prewarmed amino acid–free medium at 36°C. We found that the temperature shift was sufficient to inhibit delivery of newly synthesized protein to the plasma membrane because no increase in Gap1p activity was observed after transfer to amino acid–free medium (Figure 4A). When amino acids were added to sec6-4 strains expressing Gap1pK9R,K16R, we also observed a rapid decrease in Gap1p activity (Figure 4A), even though this nonubiquitinated form of Gap1p was not internalized (Figure 2, A and B). Most importantly, the activity of Gap1pK9R,K16R regenerated after amino acids were washed from the medium even at the restrictive temperature for sec6-4, a condition that blocks Gap1p delivery to the plasma membrane by exocytosis (Figure 4A). The conclusion from these experiments is that inactivation of Gap1pK9R,K16R is reversible in the sense that permease that has been inactivated in the plasma membrane by the addition of exogenous amino acids will recover almost full activity once amino acids are withdrawn from the medium.
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bul2) mutations that affect Gap1p ubiquitination were unable to grow in the presence of 1 mM glycine (Figure 5A). This inhibitory effect was not specific to glycine because 3 mM addition of any amino acid except alanine or phenylalanine greatly inhibited the growth of GAP1K9R,K16R (Table 2). Amino acid addition both inhibited cell growth and was cytotoxic, because less than 1% of Gap1pK9R,K16R-expressing cells were able to form colonies on medium lacking amino acids after incubation in 3 mM glycine for 22 h (Table 2).
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strain showed no effect on growth after treatment with glycine, indicating that the transient growth inhibition of wild-type strains was due to amino acid import through Gap1p (Figure 6C). Therefore, it appears that the ability of wild-type Gap1p to be efficiently ubiquitinated prevents wild-type cells from fully succumbing to amino acid induced toxicity (Chen and Kaiser, 2002). From our findings, we determined that the regulation of Gap1p sorting in response to amino acids is required to protect cells from uptake of amino acids to lethal levels.
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bul2 gap1 strain and screened for mutants that were resistant to 4 mM citrulline, a concentration that is toxic to cells expressing wild-type Gap1p in a bul1 bul2 background. Citrulline-resistant clones were then counterscreened for sensitivity to 1 mM glycine, to eliminate mutants that had lost Gap1p activity altogether. Five GAP1 alleles that conferred resistance to citrulline, but sensitivity to glycine, were isolated. Four mutants contained single, but unique point mutations: Gap1pL185V, Gap1pA297V, Gap1pV363G, and Gap1pA497V, and one mutant contained two unique mutations: Gap1pA365V,T590A. It was determined that each of these mutants retained the ability to transport [14C]glycine although ability to import [14C]citrulline was impaired to various extents (Figure 9A).
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Wild-type cells import [14C]arginine through Gap1p as well as the arginine-specific permease Can1 (Grenson et al., 1966; Whelan et al., 1979). Indeed, we found that although gap1 or can1 mutants import arginine, the double gap1 can1 mutant could not (Figure 9B). Similarly, when Gap1pK9R,K16R,A297V was the sole form of Gap1p expressed in a can1 mutant, the strain was not able to import [14C]arginine, indicating that Gap1pK9R,K16R,A297V was defective for arginine import (Figure 9B). When we measured the ability of arginine to inactivate [14C]glycine import, we found Gap1pK9R,K16R,A297V activity was unaffected by arginine, whereas Gap1pK9R,K16R activity dropped to less than 5% after an hour of arginine addition (Figure 9C). Because arginine is imported into both strains through the Can1 permease, the inability of Gap1pK9R,K16R,A297V to be inactivated upon arginine addition indicates that elevated external or internal arginine levels are insufficient to inactive the permease. Therefore, the amino acid–dependent inactivation of Gap1p at the plasma membrane must require active transport of amino acids through the permease.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Stanbrough et al., 1995; Chen and Kaiser, 2002). Here we describe a third mode of regulation: activity-dependent, reversible inactivation of permease activity at the plasma membrane. The key experiment that demonstrates inactivation is based on a mutant form of Gap1p (Gap1pK9R,K16R) that is constitutively expressed and trafficked to the plasma membrane. When amino acids are added to cells expressing Gap1pK9R,K16R, the protein loses transport activity while remaining located in the plasma membrane. This inactivation is distinct from the well-documented competitive inhibition of Gap1p transport of one amino acid by a different amino acid (Woodward and Cirillo, 1977; Woodward and Kornberg, 1981). Competitive inhibition occurs essentially instantaneously but requires the presence of the competing amino acid during the assay, whereas the inactivation we observe occurs with a half-time of 20 min and can be assayed in the absence of a competing amino acid.
Two additional observations provide further insight into the mechanism of inactivation. Amino acids do not appear to inactivate the permease irreversibly, because after withdrawal of exogenous amino acids Gap1pK9R,K16R located in the plasma membrane regains activity with a half-time of 20 min. Moreover, by evaluating a mutant of Gap1p that is defective for transport of arginine but not glycine, we found that inactivation requires active amino acid transport through the Gap1p permease. From measurements of initial amino acid import rates and the half-time required for inactivation we estimate that an individual Gap1p permease is able to transport 5000 amino acid molecules before being inactivated. Together these results imply that inactivation involves some kind of reversible modification or conformation that occurs as part of the catalytic cycle.
It has previously been shown that Gap1p is de-phosphorylated upon glutamine addition to low-phosphate urea medium or upon ammonia addition to proline medium (Stanbrough and Magasanik, 1995; De Craene et al., 2001). We however failed to observe any change in permease mobility associated with the amino acid–dependent inactivation or reactivation of the Gap1pK9R,K16R by SDS-PAGE, suggesting that protein phosphorylation may not be involved in this process (unpublished data). Additionally, it has been speculated that the manganese transporter, Smf1p, adopts a conformational change when bound to metal that influences trafficking of the permease, because transport-deficient Smf1p mutant proteins are unable to be redirected to the plasma membrane from internal compartments upon metal starvation (Liu and Culotta, 1999). It is possible that a similar conformational change occurs to Gap1p upon amino acid transport that would alter the structure of the pore to impair further amino acid import. Additional mutational analysis and biochemical characterization of the Gap1p permease will be required to elucidate the exact mechanism of permease inactivation at the plasma membrane.
Interestingly, a similar type of substrate-induced inactivation may be involved in regulation of GLAST, a neuronal sodium-dependent glutamate transporter. GLAST is highly expressed in glial cells where it takes up extracellular glutamate and thus attenuates glutamate signaling between surrounding neurons (Gonzalez and Robinson, 2004). Conditions that cause low GLAST-dependent glutamate import cause abnormally high extracellular glutamate levels leading to neuronal cell death via excitotoxicity (Huguenard, 2003). Preincubation of glial cells with glutamate or other transportable agonists can lead to a marked decrease in GLAST activity if sodium is present during the preincubation period, indicating that inactivation requires active glutamate transport (Gonzalez and Ortega, 2000). It is further suggested that glutamate affects GLAST activity by altering the affinity and not the level of permease present at the plasma membrane as changes in the Km but not Vmax of GLAST activity are observed after glutamate preincubation (Gonzalez and Ortega, 2000). Just as amino acids can regulate Gap1p activity by a variety of different mechanisms, glutamate not only inactivates GLAST permease in the plasma membrane, but also may influence the transcription and intracellular trafficking of GLAST (Lopez-Bayghen et al., 2003; Gonzalez and Robinson, 2004). The ability to study substrate induced, transport-dependent permease inactivation in a genetically tractable yeast system should allow studies to elucidate a general understanding of the process of permease inactivation at the plasma membrane.
Our work with Gap1pK9R,K16R also led to the surprising observation that cells expressing this hyperactive form of Gap1p are sensitive to addition of amino acids to the growth medium. Because these cells are more sensitive to individual amino acids than mixtures of amino acids, we deduce that toxicity results from alterations in internal amino acid pools brought about by rapid uptake and internal accumulation of a single amino acid before the permease can be inactivated.
We considered the possibility that an overabundance of one amino acid may indirectly cause amino acid starvation by feedback regulation of amino acid biosynthetic pathways. To test this idea we used GCN4 as a reporter for amino acid starvation (Hinnebusch, 2005). Previous studies showed that elevated internal levels of one amino acid can induce starvation for other amino acids; this starvation results in elevated GCN4 expression (Niederberger et al., 1981; Hinnebusch, 1984). However, we saw no increase in GCN4 expression upon the addition of individual amino acids to GAP1K9R,K16R, suggesting that the growth inhibition seen under these conditions is not a consequence of amino acid starvation (unpublished data). As a control we found that 3-aminotriazole (a competitive inhibitor of histidine biosynthesis) induced GCN4 expression in GAP1K9R,K16R.
Another possibility we considered was that highly skewed internal amino acid pools could lead to frequent amino acid misincorporation into proteins. To test this idea, we pretreated GAP1K9R,K16R with cycloheximide to block all new protein synthesis before amino acid addition. Indeed, we found a three- to fivefold increase in viability of Gap1pK9R,K16R expressing cells treated with cycloheximide before lysine addition (unpublished data). Although this finding suggests protein synthesis may play a role in the sensitivity of cells to individual amino acid addition, it is important to note that 90% of cells succumbed to toxicity, even with cycloheximide treatment, indicating that the cause of toxicity may be more complex. We also used Hsp104 as a reporter for global protein misfolding (Sanchez et al., 1992; Trotter et al., 2002). When single amino acids were added to GAP1K9R,K16R, we failed to observe a increase in Hsp104 expression similar to that seen for misincorporated amino acid analogs such as L-azetidine-2-carboxylic acid (unpublished data). Taken together these data suggest that tRNA synthetase mischarging and protein misfolding may play a role in amino acid induced toxicity.
Intracellular sorting of Gap1p appears to have evolved as a feedback mechanism to adjust Gap1p activity according to the quantity of amino acids in the cytoplasm. We propose that when internal amino acids are scarce, the high-affinity, low-specificity Gap1p permease acts as an efficient scavenger of amino acids in the extracellular environment. When cells encounter conditions of elevated external amino acids, transport of amino acids by Gap1p causes both inactivation of the permease at the plasma membrane and triggers sorting of newly synthesized Gap1p protein to the vacuole. Both effects act to limit the uptake of potentially toxic quantities of extracellular amino acids. Meanwhile, the cell can continue to take advantage of the nutrient-rich situation without the threat of toxicity through the SSY1-dependent induction of low-affinity permeases that allow for a more controlled uptake of amino acids when they are readily available in the external medium. In this manner the cell can take full advantage of conditions ranging from amino acid shortage to abundance.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020. 
Address correspondence to: Chris A. Kaiser (ckaiser{at}mit.edu)
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), and an equal volume of SD was added to the other (
). Cells were allowed to grow at 30°C, and growth was measured by optical density for 2 h after addition.
). Cells were allowed to grow at 30°C, and growth was measured by optical density. (B) Glycine, 1 mM, was added to a wild-type strain (CKY443), and Gap1p activity was measured by [14C]citrulline uptake at the indicated time after glycine addition. Three independent measurements were averaged. (C) Performed as in A with gap1
