Volatile Anesthetics Affect Nutrient Availability in Yeast

Corresponding author: Ralph L. Keil, The Milton S. Hershey Medical Center, The Pennsylvania State University, 500 University Dr., Hershey, PA 17033., rkeil{at}psu.edu (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Volatile anesthetics affect all cells and tissues tested, but their mechanisms and sites of action remain unknown. To gain insight into the cellular activities of anesthetics, we have isolated genes that, when overexpressed, render Saccharomyces cerevisiae resistant to the volatile anesthetic isoflurane. One of these genes, WAK3/TAT1, encodes a permease that transports amino acids including leucine and tryptophan, for which our wild-type strain is auxotrophic. This suggests that availability of amino acids may play a key role in anesthetic response. Multiple lines of evidence support this proposal: (i) Deletion or overexpression of permeases that transport leucine and/or tryptophan alters anesthetic response; (ii) prototrophic strains are anesthetic resistant; (iii) altered concentrations of leucine and tryptophan in the medium affect anesthetic response; and (iv) uptake of leucine and tryptophan is inhibited during anesthetic exposure. Not all amino acids are critical for this response since we find that overexpression of the lysine permease does not affect anesthetic sensitivity. These findings are consistent with models in which anesthetics have a physiologically important effect on availability of specific amino acids by altering function of their permeases. In addition, we show that there is a relationship between nutrient availability and ubiquitin metabolism in this response.

THE initial public demonstration of general anesthesia in 1846 marked a pivotal event in the history of medicine. Until that time, surgery was performed only as a last, desperate resort because of the unbearable pain and suffering inflicted on the patient. Although volatile anesthetics are essential for modern clinical practice due to their ability to render patients unconscious and insensitive to pain, the mechanisms and sites of action of these drugs remain unknown.

Volatile anesthetics affect all cells and tissues that have been tested, including a wide array of mammalian neuronal and nonneuronal cells, plant cells, yeast, and bacteria (OVERTON 1901 ; KEIL et al. 1996 ; BATAI et al. 1999 ). We are taking a molecular genetic approach to investigate anesthetic action using the yeast Saccharomyces cerevisiae. We find that volatile anesthetics arrest yeast cell division in a manner that strikingly parallels the activities of these drugs in mammals (KOBLIN 1994 ; KEIL et al. 1996 ; WOLFE et al. 1998 ). These parallels include correlation of lipophilicity and potency (the Meyer-Overton rule; KOBLIN 1994 ); rapid and reversible effects; a sharp dose-response curve; additivity of partial doses of different anesthetics; and volatile, lipophilic compounds that do not induce anesthesia in mammals do not inhibit growth of yeast. These similarities indicate that the manner in which anesthetics inhibit yeast growth and induce mammalian anesthesia may be closely related, if not identical.

To investigate anesthetic action in S. cerevisiae, spontaneous mutants that confer resistance to the growth-inhibitory effects of the volatile anesthetic isoflurane have been isolated and characterized (KEIL et al. 1996 ; WOLFE et al. 1998 , WOLFE et al. 1999 ). Two of the genes identified in this analysis have been implicated in ubiquitin metabolism: ZZZ1 is identical to BUL1, which encodes a protein that interacts with yeast ubiquitin ligase (YASHIRODA et al. 1996 ), and ZZZ4 is identical to DOA1, which affects degradation of ubiquitinated proteins (GHISLAIN et al. 1996 ). Finding that mutations in these genes affect anesthetic sensitivity suggests that ubiquitin metabolism plays a critical role in the normal response of yeast to these drugs.

To identify additional proteins involved in yeast anesthetic response, we isolated genes that, when present in multiple copies, confer resistance to the volatile anesthetic isoflurane. Here we report that one of these genes, WAK3, is identical to TAT1, which encodes a yeast amino acid permease (SCHMIDT et al. 1994 ). Finding that overexpression of an amino acid permease confers anesthetic resistance suggests that nutrient availability may be a critical factor in yeast anesthetic response. Multiple, mutually supportive experimental findings are consistent with this proposal. In addition, we find a relationship between ubiquitin metabolism and nutrient availability in the response of yeast to these drugs.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and anesthetic exposure:
Yeast strains used in this study are derivatives of RLK88-3C (LIN and KEIL 1991 ) and are listed in Table 1. Strains P1353, P1337, and P1361 were constructed from RLK88-3C by PCR-directed correction of the chromosomal leu2-3,112 and/or trp1-HIII mutations. Oligonucleotides used to generate the LEU2 and TRP1 PCR products from plasmid-borne copies of the wild-type genes were as follows: LEU2, 0-230 (5'-GAATACTCAGGTATC-3') and 0-231 (5'-TCGTAAGGCCGTTTC-3'); and TRP1, 0-228 (5'-GTGACTATTGAGCAC-3') and 0-229 (5'-GTGCACAAACAATAC-3'). PCR products were transformed into RLK88-3C, and stable Leu⁺ or Trp⁺ transformants were isolated. In a similar manner, strain P1814 was constructed from P1361 by PCR-directed correction of the chromosomal his4-260 and ade2-1 mutations. Oligonucleotides used to generate the HIS4 and ADE2 PCR products from plasmid-borne copies of the wild-type genes were as follows: HIS4, 0-253 (5'-TCATCGGAAGAGGTGGCATC-3') and 0-254 (5'-AAACCGTCAGGACGGTCTGT-3'); and ADE2, 0-259 (5'-GAAACTTCATGCTCGAAAAAG-3') and 0-260 (5'-CAAGGGAACATTATAGGGTG-3'). Unless otherwise noted, yeast (LIN and KEIL 1991 ) and bacterial (SAMBROOK et al. 1989 ) media were prepared as previously described. Isoflurane (Baxter Healthcare Corporation) exposure of yeast grown on solid media was performed as described previously (KEIL et al. 1996 ; WOLFE et al. 1999 ).

Isolation of anesthetic resistance-conferring genes:
To identify genes that confer resistance to anesthetics when present in multiple copies, RLK88-3C was transformed (SCHIESTL and GIETZ 1989 ) with a 2µ-based (YEp; multicopy) yeast genomic plasmid library (CARLSON and BOTSTEIN 1982 ) and screened for transformants resistant to isoflurane. Plasmids recovered from these transformants were propagated in Escherichia coli strain MC1066 [leuB trpC pyrF::Tn5 (Kan^r) araT lacX74 del strA hsdR hsdM (obtained from M. Casadaban)].

DNA manipulations, plasmids, and gene deletions:
PCR reagents as well as restriction and modification enzymes were purchased from various sources and used according to the instructions of the manufacturers. Standard procedures for the purification of plasmid (SAMBROOK et al. 1989 ) and yeast genomic (ROSE et al. 1990 ) DNA were used.

Plasmid pL3271 contains an 11.1-kb fragment of yeast genomic DNA from chromosome II that includes WAK3. This fragment is inserted in the BamHI site of YEp24 (BOTSTEIN et al. 1979 ). Oligonucleotides used to sequence into the insert from both ends were 0-73 (5'-GCCAGCAACCGCACC-3') and 0-74 (5'-GCCACTATCGACTAC-3'), which hybridize to plasmid sequences flanking the insert. DNA sequencing was performed in the Molecular Genetics Core Facility of the M. S. Hershey College of Medicine using an ABI 377 DNA sequencer.

To initially localize the sequences encoding WAK3, deletion derivatives of pL3271 were constructed by digestion with convenient restriction enzymes. The restricted DNA was religated to produce plasmids with the various deletions. Among the deletion derivatives constructed were pL3273, which contains the C-terminal portion of TAT1 and the entire protein-encoding sequence for BAP2, and pL3277, which contains the entire TAT1 gene and the amino-terminal portion of BAP2. These plasmids are termed YEpBAP2 and YEpTAT1, respectively.

TAT1 and TAT2 on the low-copy YCplac33 vector (GIETZ and SUGINO 1988 ), named YCpTAT1 and YCpTAT2, respectively (original designations pTAT1 and pTAT2; SCHMIDT et al. 1994 ), were kindly provided by M. Hall. The 3.7-kb EcoRI fragment from YCpTAT2 containing the TAT2 open reading frame and flanking genomic sequences was ligated into the EcoRI site of the URA3-marked YEplac195 vector (GIETZ and SUGINO 1988 ). This plasmid was termed YEpTAT2. The high-affinity lysine permease, LYP1, on the 2µ-based pYX212 vector (original designation pRB165; REGENBERG et al. 1999 ), and BAP2 on the pYX212 vector (original designation pRB145; REGENBERG et al. 1999 ) were obtained from M. C. Kielland-Brandt. These plasmids were termed YEpLYP1 and YEpBAP2-2, respectively.

The entire protein-encoding sequences of TAT1 or BAP2 were deleted from RLK88-3C and replaced with loxP-kanMX-loxP from pUG6 (GULDENER et al. 1996 ) by using appropriate PCR-generated gene disruption cassettes. Oligonucleotides used to generate these cassettes were as follows: TAT1, 0-153 (5'-GTAGCTACCTAATATAGTTTCTCGATAAAAAGGCGTAAAACAGGTCGACAACCCTTATA-3') and 0-154 (5'-AAGCCCGATGAAGCCAAGCGGAAAATGAATCGAATTGCTGGTGGATCTGATATCACCTA-3'); and BAP2, 0-216 (5'-CAATTTATTTAGCTTCAATAAAACTCAAGTGATTTTAGAACAGGTCGACAACCCTTAAT-3') and 0-217 (5'-TCTAATGGGTAGTGTCCAGACCTGAGTGGTGTAGTTAAGTGTGGATCTGATATCACCTA-3'). Correct gene deletions were verified by PCR.

To delete the entire protein-encoding sequence of TAT2 in RLK88-3C, a vector derived from YCpTAT2 in which the TAT2 gene was precisely replaced with a loxP-kanMX-loxP fragment was constructed as follows: Inverse PCR was used to create a derivative of YCpTAT2 in which the TAT2 gene was precisely deleted and replaced with a NotI restriction site. Oligonucleotides 0-384 (5'ATACGATAGCGGCCGCATGAGAGTGTGTTGCGTAATTTGC-3') and 0-385 (5'-ATAAGATAGCGGCCGCTACCGAAGAAACAAGTTC-3') were used for this PCR reaction. The resulting PCR product was digested with NotI and the 1.6-kb NotI fragment of pUG6 containing loxP-kanMX-loxP was inserted, producing pL4071. A PCR product was generated from pL4071 using oligonucleotides 0-373 (5'-CATGATATTGCATC TACCTC-3') and 0-374 (5'-AAATTTGATTCCTACGGCAG-3') and transformed into RLK88-3C. The tat2::loxP-kanMX-loxP transformants created by this process were identified on the basis of resistance to G418, and the occurrence of the appropriate deletion was verified by PCR.

Amino acid import studies:
To measure leucine or tryptophan uptake in the presence or absence of isoflurane, cells were grown to an approximate OD₆₀₀ of 0.4 in synthetic complete (SC) media (LIN and KEIL 1991 ) containing 70 µg/ml leucine and 10 µg/ml tryptophan. Twenty-five-milliliter aliquots of this culture were injected into 250-ml evacuated bottles (Baxter Healthcare Corporation) containing the desired concentration of volatilized anesthetic. Air was admitted into the bottles to achieve 1 atmosphere of pressure. After 15 min of incubation at 30°, 20 µCi of L-[U-¹⁴C]leucine (50 µCi/ml; Amersham, Arlington Heights, IL) or 80 µCi of L-[5-³H]tryptophan (1 mCi/ml; Amersham) was added. Triplicate samples of cells were removed at 0- and 10-min time intervals after the addition of the labeled amino acid, collected on glass-fiber filters, and washed extensively with cold liquid medium containing excess unlabeled leucine (20 mg/ml) and tryptophan (4 mg/ml). Washed filters were placed in scintillation vials and treated with 1 M NaOH. This solution was neutralized with 1 M acetic acid and liquid scintillation fluid (Fisher Scientific, Pittsburgh) was added. Uptake of labeled amino acid into the cells was determined by liquid scintillation counting and levels in the presence or absence of isoflurane were compared.

To measure leucine uptake in tat1, YEpTAT1, zzz1, or zzz4 strains or lysine uptake in RLK88-3C transformed with YEpLYP1 or the vector control, 25-ml aliquots of cells grown to an approximate OD₆₀₀ of 0.4 in SC or SC-ura medium were harvested and resuspended in 1.25 ml of fresh media. The cell suspension was added to SC or SC-ura medium containing 1.25 µCi of L-[U-¹⁴C]leucine or 5 µCi of L-[4,5-³H]lysine (1 mCi/ml; Amersham) to a total volume of 3.2 ml. Triplicate samples of cells were removed at 0- and 10-min time points after addition of the cells to the medium containing the labeled amino acid and collected on glass-fiber filters. The filters were washed and treated as described above, and radioactivity was quantified by liquid scintillation counting.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Altered levels of TAT1 affect yeast anesthetic response:
To identify novel genes involved in volatile anesthetic action in S. cerevisiae, an overexpression library containing random fragments of yeast genomic DNA inserted into the 2µ-based YEp24 vector (BOTSTEIN et al. 1979 ) was screened for plasmids that confer resistance to the volatile anesthetic isoflurane. Seven transformants resistant to a normally growth-inhibitory concentration of isoflurane [12%; the minimum inhibitory concentration (MIC; KEIL et al. 1996 )] were isolated from 20,000 transformants. Loss of the plasmids from these transformants resulted in reversion to the anesthetic-sensitive phenotype of the wild-type strain, RLK88-3C (Table 1), indicating that the anesthetic resistance was due to plasmid-borne genes. Plasmids from each of these transformants were recovered into E. coli and reintroduced into RLK88-3C. All of the transformants tested were anesthetic resistant, further demonstrating that the plasmids were responsible for altering anesthetic response. Genes on these plasmids critical for anesthetic resistance (identified in subsequent analyses) were termed WAK (pronounced "wake") genes to indicate their roles in preventing yeast growth inhibition, or "sleep," produced by anesthetic agents.

Restriction mapping of the plasmids indicated that five of the seven plasmids contained a common DNA segment. These plasmids represent five isolates containing the WAK3 gene. Sequencing into the insert in one of these plasmids, pL3271, revealed that the insert was an 11.1-kb DNA fragment from chromosome II containing the carboxy terminus of YBR070C, full-length TAT1, BAP2, TIP1, and NRG2 genes, and the amino-terminal portion of ECM2 (Fig 1). Deletion analysis of pL3271 showed that TAT1 was essential for producing the anesthetic resistance (Fig 1 and Fig 2). TAT1 encodes an amino acid permease that transports leucine, tryptophan, isoleucine, valine, and tyrosine (SCHMIDT et al. 1994 ; REGENBERG et al. 1999 ). Even when present on a low-copy, centromeric plasmid (YCplac33; GIETZ and SUGINO 1988 ), TAT1 increased the anesthetic resistance of RLK88-3C (Fig 2, compare strains 5 and 6), indicating that a relatively small increase in TAT1 expression is sufficient to alter anesthetic response. These results suggest that nutrient availability may be a critical determinant in the anesthetic response of yeast.

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. WAK3 and flanking genomic DNA. WAK3 was identified on the basis of deletion analysis of an 11.1-kb fragment of yeast genomic DNA contained in plasmid pL3271. Thick lines represent multicopy vector (YEp24) sequences and thin lines represent the fragment of yeast genomic DNA containing WAK3. The horizontal arrows indicate the open reading frames contained within this fragment and their direction of transcription, with the arrowhead at the 3' terminus. The short dashed vertical lines indicate the positions at which the YBR070C and ECM2 genes are truncated in pL3271. The sequences present in several deletion derivatives of this plasmid are shown below the restriction map of the fragment, with the lines indicating the DNA present in the deletion derivative. Strain numbers refer to strains shown in Fig 2 that contain the indicated plasmid derivatives; NS, not shown in Fig 2. The level of isoflurane resistance (IsoR) of each derivative as compared to the wild-type strain (RLK88-3C; Table 1) is indicated: -, same level of resistance as the wild-type strain; ++, intermediate level of isoflurane resistance; +++, higher level of isoflurane resistance.

View larger version (91K): In this window In a new window Download PPT slide   Figure 2. Increased levels of TAT1 affect isoflurane MIC. Approximately 104 cells from freshly saturated cultures of the indicated strains were spotted on SC-ura medium and incubated for 3 days at 30° in the presence or absence of various concentrations of isoflurane (Iso). YEp, multicopy plasmid containing the indicated permease gene(s); YCp, low-copy-number plasmid containing the indicated permease gene.

In addition to Tat1p, pL3271 also contains the protein-encoding sequence for Bap2p, a branched-chain amino acid permease that transports leucine, isoleucine, and valine (GRAUSLUND et al. 1995 ). Although deletion analysis clearly showed that TAT1 was indispensable for the anesthetic resistance, a plasmid containing both TAT1 and BAP2 rendered RLK88-3C more resistant to isoflurane than did a plasmid overexpressing only TAT1 (Fig 2, compare strains 2 and 4). Overexpression of BAP2 alone did not render cells anesthetic resistant (Fig 2, compare strains 1 and 3), suggesting that while increasing the availability of only branched-chain amino acids is not sufficient to impart anesthetic resistance to RLK88-3C, it can augment the level of resistance provided by overexpression of TAT1.

If overexpression of TAT1 confers anesthetic resistance by increasing amino acid availability, decreased levels of this permease might render cells hypersensitive to anesthetics due to reduced ability of the cells to transport one or more critical amino acids. To test this possibility, the anesthetic response of cells containing a precise deletion of the protein-encoding sequence of TAT1 was examined. We find that deletion of TAT1 increases the sensitivity of our wild-type strain (Fig 3, compare strain 1 TAT1 to strain 1 tat1). This provides further evidence that TAT1 plays a critical role in anesthetic response.

View larger version (79K): In this window In a new window Download PPT slide   Figure 3. Deletion of TAT1 (tat1) decreases isoflurane resistance of Leu- or Trp- strains but not of a Leu+Trp+ strain. Approximately 104 cells from freshly saturated cultures of the indicated strains were spotted on SC medium and tested for response to isoflurane (Iso).

Other permeases can affect anesthetic response:
Of the amino acids transported by Tat1p, our wild-type strain is auxotrophic for only leucine and tryptophan (Table 1), suggesting that the availability of one or both of these amino acids affects anesthetic response. The finding that BAP2 does not confer anesthetic resistance to RLK88-3C when overexpressed alone raises the possibility that TAT1 is unique in its ability to alter anesthetic response. Alternatively, the ability of TAT1 to transport both leucine and tryptophan may be the critical property, and permeases that transport only one of these amino acids may have the ability to alter anesthetic response if overexpressed in appropriately prototrophic strains. To distinguish between these possibilities, anesthetic response was assessed when BAP2 was overexpressed in yeast strains with varying leucine or tryptophan prototrophies. Overexpression of BAP2 in a Leu^-Trp^- strain had no effect on the isoflurane MIC (Fig 2, compare strains 1 and 3) and only very slightly increased resistance in a Leu⁺Trp^- strain (Fig 4, compare strains 1 and 2). However, BAP2 overexpression in a Leu^-Trp⁺ derivative, which is slightly more resistant to isoflurane than the Leu^-Trp^- strain (Fig 3, compare strains 1 and 3), increased the resistance of this strain to the same level as that of overexpression of Tat1p (Fig 4, compare strains 4 and 5 to strain 3). YEpBAP2 contains open reading frames in addition to BAP2 (Fig 1, strain 3 plasmid). To ensure that the altered anesthetic response required only overexpression of BAP2, we tested a plasmid containing only BAP2 (YEpBAP2-2). We observed a similar level of resistance with this plasmid as with YEpBAP2 in the Leu^- Trp⁺ strain (data not shown). These results indicate that overexpression of BAP2, which encodes a high affinity leucine transporter, can increase anesthetic resistance when overexpressed in a strain that is prototrophic for tryptophan but auxotrophic for leucine. In a similar manner, overexpression of TAT2, which encodes the high affinity yeast tryptophan transporter, in Leu^-Trp^- or Leu^-Trp⁺ strains did not increase anesthetic resistance (Fig 4, compare strains 6 and 7 or strains 8 and 9), while overexpression of this permease in a Leu⁺Trp^- strain resulted in increased resistance to isoflurane (Fig 4, compare strains 10 and 11).

View larger version (61K): In this window In a new window Download PPT slide   Figure 4. Overexpression of (A) BAP2 or (B) TAT2 can alter anesthetic response when expressed in strains with appropriate prototrophies. Approximately 104 cells from freshly saturated cultures of strains transformed with the indicated plasmids were spotted on SC-ura medium and examined for growth in the presence of various concentrations of isoflurane (Iso). YEp, multicopy plasmid containing the indicated permease gene.

Analogous to TAT1, deletion of BAP2 or TAT2 increases the anesthetic sensitivity of RLK88-3C (Fig 5). Taken together, these results indicate that the availability of leucine and tryptophan together plays a role in anesthetic response. They also demonstrate that Tat1p is not unique in its ability to alter anesthetic response in yeast.

View larger version (69K): In this window In a new window Download PPT slide   Figure 5. Deletion of BAP2 (bap2) or TAT2 (tat2) from the Leu-Trp- wild-type strain (RLK88-3C; Table 1) decreases MIC. Approximately 104 cells from freshly saturated cultures of the indicated strains were spotted on SC medium and tested for response to isoflurane (Iso).

Overexpression of LYP1 does not affect anesthetic response:
To test whether all amino acid permeases are capable of affecting anesthetic response, we examined the behavior of a derivative of our wild-type strain that contained a multicopy plasmid encoding the high affinity lysine-specific permease, LYP1 (SYCHROVA and CHEVALLIER 1993 ). Although our wild-type strain is auxotrophic for lysine, LYP1 overexpression had no effect on the anesthetic response of this strain (Fig 6A). To verify that overexpression of LYP1 increased lysine import in this strain, uptake of radiolabeled lysine was measured. An 2.5-fold increase in lysine uptake was observed in cells overexpressing LYP1 (YEpLYP1) compared with cells containing the vector alone (YEpLYP1, 249 ± 23%; YEp, 100%).

View larger version (54K): In this window In a new window Download PPT slide   Figure 6. Lysine availability does not affect anesthetic sensitivity. (A) Overexpression of the lysine-specific permease, LYP1, does not affect the anesthetic response of RLK88-3C. Approximately 104 cells from freshly saturated cultures of RLK88-3C (his4 leu2 ura3 ade2 trp1 lys2) transformed with YEp or YEpLYP1 plasmids were spotted on selective media and tested for the ability to grow in various concentrations of isoflurane (Iso). (B) Overexpression of LYP1 inhibits growth of P1814 (HIS4 LEU2 ura3 ADE2 TRP1 lys2). P1814 strains transformed with YEp or YEpLYP1 plasmids were streaked on selective medium and incubated at 30° in the absence of anesthetic for 48 hr. (C) LYP1 overexpression does not increase the anesthetic resistance of P1814.Approximately 104 cells from freshly saturated cultures of P1814 transformed with YEp or YEpLYP1 plasmids were spotted on selective media and tested for response to isoflurane (Iso).

It is possible that LYP1 overexpression may alter anesthetic response in a strain with appropriate prototrophies, similar to the finding that overexpression of BAP2 or TAT2 only increases anesthetic response in strains that are Trp⁺ or Leu⁺, respectively. To test this possibility, a derivative of the wild-type strain that is auxotrophic only for lysine and uracil (Lys^-Ura^-) was constructed (Table 1). This strain was transformed with the URA3-marked multicopy LYP1 plasmid or the vector control and anesthetic response was assessed. Although overexpression of LYP1 decreased growth of this strain for an unknown reason even in the absence of isoflurane (Fig 6B), we find that overexpression of LYP1 does not increase anesthetic resistance (Fig 6C). Thus, lysine availability from the medium does not affect anesthetic response even when the strain is auxotrophic for only this amino acid. This indicates that Lyp1p levels are not a critical factor in yeast anesthetic response.

Leucine and tryptophan phenotypes affect anesthetic response:
To verify that the ability of some amino acid permeases to alter anesthetic response is due to their ability to transport nutrients into the cell and not some other function of the permease, anesthetic response was assessed in a series of Leu⁺ and/or Trp⁺ derivatives (Table 1) of the Leu^-Trp^- strain. We find slight increases in the level of isoflurane resistance of the Leu⁺Trp^- and Leu^-Trp⁺ derivatives as compared to the Leu^-Trp^- strain (Fig 7A, compare strains 2 and 3 to strain 1). In contrast, the isoflurane MIC of a Leu⁺Trp⁺ derivative increased from 12% to >14% (Fig 7A, compare strains 1 and 4), indicating that cells that can synthesize both leucine and tryptophan are more anesthetic resistant than cells that are auxotrophic for one or both amino acids. Deletion of TAT1 did not affect the isoflurane MIC of the Leu⁺Trp⁺ strain (Fig 3, compare strain 4 TAT1 to strain 4 tat1). This provides additional evidence that the role of Tat1p in anesthetic response is likely due to its ability to import leucine and tryptophan from the external environment.

View larger version (40K): In this window In a new window Download PPT slide   Figure 7. Excess leucine and tryptophan in the growth medium render auxotrophic strains resistant to isoflurane. Approximately 104 cells from freshly saturated cultures of the indicated strains were spotted on media containing (A) our normal concentrations of leucine and tryptophan (1x Leu 1x Trp), (B) a threefold excess of leucine (3x Leu 1x Trp), (C) a threefold excess of tryptophan (1x Leu 3x Trp), or (D) a threefold excess of both amino acids (3x Leu 3x Trp) and incubated for 3 days at 30° in the presence or absence of various concentrations of isoflurane (Iso). A strain containing a genomic deletion of ZZZ1/BUL1 (Leu-Trp- zzz1) was included as an anesthetic-resistant control.

If the ability of a cell to import amino acids is affected by volatile anesthetics, increasing the concentrations of these nutrients in the medium may alleviate the growth-inhibitory effects of these drugs. A similar approach was employed to demonstrate that excess tryptophan in the growth medium can mitigate the toxicity of the immunosuppressive drug FK506, which inhibits amino acid import in yeast (HEITMAN et al. 1993 ). To test whether excess amino acids increase anesthetic resistance, the isoflurane MICs of the Leu^-Trp^-, Leu⁺Trp^-, Leu^-Trp⁺, and Leu⁺Trp⁺ strains grown on SC medium containing a threefold excess of leucine (3x Leu 1x Trp), tryptophan (1x Leu 3x Trp), or both amino acids (3x Leu 3x Trp) were compared to those of strains grown on media containing our normal concentrations of these amino acids (1x Leu 1x Trp). While increasing the leucine concentration threefold in the medium had no effect on the isoflurane MIC of the Leu^-Trp^- or Leu⁺Trp^- strain (Fig 7B, strains 1 and 2), the MIC of the Leu^-Trp⁺ strain increased to levels similar to the prototrophic Leu⁺Trp⁺ strain (Fig 7B, compare strains 3 and 4). Increasing the tryptophan concentration threefold in the medium moderately increased the isoflurane resistance of the Leu^-Trp^- and Leu^-Trp⁺ strains, although these strains are still more sensitive than the Leu⁺Trp⁺ strain (Fig 7C, compare strains 1 and 3 to strain 4). The increased tryptophan concentration rendered the Leu⁺Trp^- strain extremely isoflurane resistant, to a level similar to that of the Leu⁺Trp⁺ strain (Fig 7C, compare strains 2 and 4). Increasing the concentrations of both leucine and tryptophan rendered all strains extremely resistant to isoflurane regardless of their leucine or tryptophan phenotypes (Fig 7D). These results further emphasize that availability of both leucine and tryptophan plays a critical role in anesthetic response and that high levels of amino acids in the medium are able to alleviate anesthetic-induced growth inhibition of strains with appropriate auxotrophies.

Anesthetics inhibit amino acid import:
The results described above suggest that anesthetics may inhibit yeast cell division by decreasing amino acid import. Thus, a reasonable explanation for the increased isoflurane resistance conferred by TAT1 overexpression is that it increases uptake of amino acids from the external environment, allowing the cell to grow in the presence of a normally inhibitory concentration of anesthetic. Indeed, we find that overexpression of TAT1 increases leucine uptake in our wild-type strain 35% (Table 2). In addition, we find an 20% decrease in leucine uptake in an anesthetic-supersensitive tat1 strain as compared to its wild-type counterpart (Table 2). Taken together, these results suggest a correlation between the level of amino acid import and the level of isoflurane resistance in yeast.

To directly test the effect of isoflurane exposure on amino acid import, transport of radiolabeled leucine or tryptophan was measured in the presence and absence of isoflurane. As shown in Table 3A, leucine import decreased 55% in anesthetic-exposed wild-type cells after 15 min of exposure to isoflurane. This rapid inhibition also corresponds to the time frame in which anesthetic-induced growth inhibition occurs (WOLFE et al. 1998 ). The observed decrease in leucine import as a result of anesthetic exposure (Table 3A) is substantially larger than the decrease observed in the tat1 strain (Table 2), which is hypersensitive to isoflurane. This is consistent with our finding that leucine permeases, such as Bap2p, in addition to Tat1p play a role in anesthetic response. Similar results were observed for tryptophan import. After 15 min of isoflurane exposure, import of this amino acid decreased 50% (Table 3A), and an even greater decrease (60%) was observed after 30 min of exposure (Table 3A), indicating that incubation for extended periods of time in isoflurane further decreases amino acid import.

Nutrient availability and ubiquitin metabolism are related in the anesthetic response of yeast:
The results from this study indicate that nutrient availability plays a critical role in the cellular response of yeast to volatile anesthetics. Our previous investigations have shown that ubiquitin metabolism also affects anesthetic response. Mutations in ZZZ1, which is identical to BUL1 (binds ubiquitin ligase), or ZZZ4, which is identical to DOA1/UFD3 and was previously identified on the basis of altered degradation of ubiquitinated proteins, render cells resistant to isoflurane (KEIL et al. 1996 ; WOLFE et al. 1999 ). Ubiquitination has been implicated in regulating the turnover and cellular distribution of amino acid permeases in yeast (HEIN et al. 1995 ; HICKE 1997 ; BECK et al. 1999 ; HELLIWELL et al. 2001 ; OMURA et al. 2001 ). These findings raise the possibility that the role of ubiquitin metabolism in anesthetic response is related to nutrient availability through regulation of amino acid permeases. To test this possibility, we measured leucine import in zzz1 and zzz4 strains. In the absence of anesthetic, we find an approximately twofold increase in the amount of leucine import in a zzz1 strain compared to its isogenic wild-type counterpart (Table 2). The ability of this anesthetic-resistant mutant to import higher levels of amino acids is further supported by the finding that it is able to grow much better than the wild-type strain on media containing decreased levels of leucine and tryptophan (Fig 8, compare strains 1 and 2). Although only a small increase in leucine import was detected in the zzz4 strain as compared to the wild-type strain (Table 2), zzz4 mutants had a slight growth advantage compared to wild-type cells when grown on media with low levels of leucine and tryptophan (Fig 8, compare strains 1 and 3 at 0.4x Leu 0.4x Trp). This suggests that only a slight increase in amino acid import is sufficient to affect anesthetic response and is reminiscent of the finding that even a single extra copy of TAT1 (YCpTAT1) can increase anesthetic resistance (Fig 2, strain 6).

View larger version (68K): In this window In a new window Download PPT slide   Figure 8. Anesthetic-resistant mutants grow better than the isogenic wild-type strain on media containing low levels of leucine and tryptophan. Approximately 104 cells from freshly saturated cultures of wild-type, zzz1, and zzz4 strains were spotted on media containing the indicated concentrations of leucine and tryptophan. Growth was evaluated following incubation at 30° for 40 hr.

We also tested the effect of isoflurane exposure on amino acid import in zzz1 cells. Leucine import decreased 50% when these cells were exposed for 15 min to a concentration of isoflurane that inhibits growth of wild-type but not zzz1 strains (Table 3B). This anesthetic-induced decrease of leucine import in the zzz1 strain is similar in magnitude to that observed in the wild-type strain (Table 3A). However, since zzz1 cells import approximately twofold more leucine in the absence of isoflurane (Table 2), the 50% reduction induced by isoflurane leads to import levels similar to that for unexposed wild-type cells (Table 3B). Thus, the ability of zzz1 strains to grow in the presence of normally inhibitory concentrations of isoflurane may be due to their continued import of sufficient levels of amino acids.

Taken together, these results establish a relationship between ubiquitin metabolism and nutrient availability in anesthetic response of yeast. The data are also consistent with our previous finding that zzz1 mutants display a much higher level of resistance to isoflurane than do zzz4 mutants (WOLFE et al. 1999 and Fig 9).

View larger version (80K): In this window In a new window Download PPT slide   Figure 9. Anesthetic MIC decreases on medium containing low levels of leucine and tryptophan. Approximately 104 cells from freshly saturated cultures of the indicated strains were spotted on medium containing our normal concentrations of leucine and tryptophan (1x Leu 1x Trp) or medium containing one-half the normal concentrations of these amino acids (0.5x Leu 0.5x Trp) and tested for response to isoflurane (Iso).

Anesthetic MIC can be decreased:
One difference in the behavior of anesthetic agents in yeast and humans is that an 10-fold higher concentration of anesthetic is required to inhibit growth in yeast than is required to anesthetize a human (KEIL et al. 1996 ). It is possible that excess amino acids in our formulation of yeast growth medium may contribute to this high level of resistance. Thus, decreasing leucine and tryptophan levels in the medium may decrease the anesthetic MIC. To test this possibility, the isoflurane phenotypes of wild-type and zzz strains were compared on media containing 1x and 0.5x concentrations of leucine and tryptophan. Decreasing the amino acid levels in the medium reduced the MIC of the wild-type strain from the normal 12% to 9% (Fig 9, compare strains 1 and 4), indicating that it is possible to manipulate the anesthetic MIC simply by altering the leucine and tryptophan composition of the medium. Lowering the amino acid levels also increased the sensitivity of the zzz4 strain (Fig 9, compare strains 3 and 6). In contrast, the MIC of the strongly anesthetic-resistant zzz1 mutant was not affected on the 0.5x Leu 0.5x Trp media (Fig 9, compare strains 2 and 5) and may require an even greater reduction in the leucine and tryptophan concentrations to observe an effect.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our results indicate that volatile anesthetics inhibit yeast cell division by affecting the availability of amino acids, in particular leucine and tryptophan for our strain, from the external environment. Numerous mutually supportive experimental findings are consistent with this statement. First, deletion or overexpression of amino acid permeases that transport leucine and/or tryptophan affects the anesthetic response of appropriately auxotrophic strains. Second, strains that are prototrophic for leucine and tryptophan are much more resistant to isoflurane than auxotrophic strains. Third, increased concentrations of leucine and tryptophan in the medium make auxotrophic strains resistant to these drugs, while decreased concentrations of these amino acids in the medium make them more sensitive. Fourth, uptake of radiolabeled leucine or tryptophan is inhibited during anesthetic exposure. These findings are consistent with models in which anesthetics affect amino acid availability by either directly or indirectly affecting amino acid permeases.

Role of amino acid permeases in anesthetic response:
One potential role for amino acid permeases is that they may behave as drug pumps similar to the ATP-binding transporters that are involved in pleiotropic drug resistance in yeast (BALZI and GOFFEAU 1995 ). In this case, increased levels of permeases on the plasma membrane (e.g., presence of YEpTAT1) would increase the export of volatile anesthetics that have entered the cell, thus decreasing the intracellular concentration of the anesthetic and rendering the cells resistant. Decreasing permease levels on the plasma membrane (e.g., deletion of TAT1) would have the opposite effect, resulting in increased anesthetic sensitivity. Although this is an intriguing possibility, our data are not readily consistent with this model. First, while deletion of TAT1 renders Leu^-Trp^-, Leu⁺Trp^-, and Leu^-Trp⁺ strains supersensitive to isoflurane, deletion in a Leu⁺Trp⁺ strain has no effect on anesthetic response (Fig 3, compare strain 4 TAT1 to strain 4 tat1). Second, overexpression of TAT1 in the Leu⁺Trp⁺ strain background also has no effect (not shown). Because the level of Tat1p is irrelevant in a strain that is prototrophic for leucine and tryptophan, this suggests that the critical property of the permease is not a generalized ability to export anesthetics out of the cell, but rather is due to its ability to import amino acids from the external environment. The findings that overexpression of the leucine transporter, BAP2, or the tryptophan transporter, TAT2, confers anesthetic resistance only in strains with appropriate prototrophies provide further evidence that the permeases are not functioning as drug export pumps. This is reminiscent of the findings of WOLFE et al. 1998 that the pleiotropic drug response genes YAP1/PDR4 and PDR5 do not affect anesthetic response.

The involvement of amino acid permeases in anesthetic response may be direct or indirect (Fig 10). Direct involvement would indicate that permeases are the primary targets of volatile anesthetics. In the absence of anesthetics, permeases would function normally, permitting the cell to grow (Fig 10A, Fig I). During anesthetic exposure, the anesthetic could bind directly to the permease and inhibit amino acid transport (Fig 10A, ii), or the anesthetic could interact with the plasma membrane near the permease, inducing a perturbation that leads to altered permease function (Fig 10A, iii). In both cases, anesthetic interaction would lead to decreased permease activity and thus inhibition of amino acid transport.

View larger version (20K): In this window In a new window Download PPT slide   Figure 10. Models for (A) direct and (B) indirect involvement of amino acid permeases in anesthetic response. See DISCUSSION for details of the models. Barrel, amino acid permease; open circles, amino acids; triangles, anesthetic; X, inhibition of amino acid transport; cross-hatched circle, post-translational modification of amino acid permease. The curved arrow indicates removal of the permease from the plasma membrane. Alternative models for the indirect involvement of amino acid permeases are possible.

An alternative explanation for decreased uptake of amino acids is that volatile anesthetics induce a post-translational modification of the permease that either directly decreases transport activity (Fig 10B, ii) or leads to altered cellular localization or degradation of the permease (Fig 10B, iii), thus affecting its activity. Our finding that mutation of a number of different ubiquitin metabolism genes affects anesthetic response (WOLFE et al. 1999 ) suggests that ubiquitination may be a potential modification. Further support for this possibility comes from the finding that a variety of plasma membrane proteins, including amino acid permeases, undergo ubiquitin-dependent endocytosis and degradation in response to environmental stimuli (HEIN et al. 1995 ; HICKE 1997 ; BECK et al. 1999 ). Of particular interest is the finding that ubiquitination and degradation of Tat2p are induced by nutrient limitation (BECK et al. 1999 ). Studies are currently in progress to determine which, if any, of these models accurately reflect the involvement of amino acid permeases in anesthetic response.

Specificity of amino acid permeases:
We find that overexpression or deletion of TAT1, BAP2, and TAT2 increases or decreases anesthetic resistance, respectively, in strains with appropriate amino acid auxotrophies. However, overexpression of LYP1 has no effect on anesthetic response. These results indicate specificity in the activity of anesthetics in yeast. In addition, it suggests that these drugs are not affecting the proton gradient necessary to drive amino acid import, as this gradient is required for transport of all amino acids. While it is not clear why the levels of some amino acid permeases affect anesthetic response while others do not, it is interesting to note that Tat1p, Bap2p, and Tat2p are more closely related evolutionarily to each other than to Lyp1p (NELISSEN et al. 1997 ). In addition, transcription of TAT1, BAP2, and TAT2 has been shown to be affected by Ssy1p (DIDION et al. 1996 ; IRAQUI et al. 1999 ), a component of a yeast plasma membrane sensor of extracellular amino acids (KLASSON et al. 1999 ; FORSBERG et al. 2001 ). Experiments are currently in progress to delineate which other amino acid permeases alter anesthetic response when their levels are genetically manipulated and to determine whether Ssy1p plays a role in anesthetic response. Results from these studies should provide insights into the nature of anesthetic specificity in yeast.

Anesthetic effects in yeast and mammals are similar:
In mammals, volatile anesthetics have been shown to dramatically affect metabolism in a variety of cells and tissues, including the brain. Of particular interest in regard to this study is the finding that anesthetics affect amino acid transport in mammalian systems. Since a number of neurotransmitters are amino acids (examples include glutamate, aspartate, and glycine) or amino acid derivatives (tryptophan is the precursor for serotonin, tyrosine is the precursor of catecholamines including dopamine, glutamate is the precursor for GABA, and histidine is the precursor of histamine), availability of amino acids and their derivatives is of critical importance for neuronal function. SHIMADA et al. 1995 showed that volatile anesthetics inhibit L-alanine transport in rat megakaryocytes (precursors of platelets). Volatile anesthetics also inhibit transport of dopamine (EL-MAGHRABI and ECKENHOFF 1993 ) and serotonin (5-hydroxytryptamine; MARTIN et al. 1990 ) in rat brain synaptosomes. In addition, while branched-chain amino acids are neither neurotransmitters nor precursors, they (especially leucine) play a major role in regulating cellular pools of the neurotransmitter glutamate (YUDKOFF et al. 1994 ; YUDKOFF 1997 ). It is intriguing that a number of these amino acids are transported by permeases that are affected by volatile anesthetics in yeast: Tryptophan and tyrosine are transported by Tat1p and Tat2p (SCHMIDT et al. 1994 ); branched-chain amino acids are transported by Tat1p and Bap2p (GRAUSLUND et al. 1995 ; NELISSEN et al. 1997 ); and Tat1p has been shown to act as a low-affinity histidine transporter (BAJMOCZI et al. 1998 ). Thus, effects of volatile anesthetics on permeases that transport any of these critical nutrients in neuronal cells as well as in other cells and tissues may play an essential role in the cellular activities of these drugs in mammals.

Biologically relevant targets:
The search for cellular targets of anesthetics that are critical for inducing anesthesia has been a predominant theme in research on these drugs for years. One standard applied in this search has been whether effects on potential targets are observed when a clinically relevant concentration of anesthetic is used. This standard has sparked a lively exchange (ECKENHOFF and JOHANSSON 1999 , ECKENHOFF and JOHANSSON 2001 ; EGER et al. 2001 ). Finding that activities of a wide range of cellular proteins are affected by clinical concentrations of anesthetics in vitro (for a review see ECKENHOFF and JOHANSSON 1997 ) makes it difficult to argue that this is an informative guidepost to distinguish between targets that are biologically significant for anesthetic action and targets that are inconsequential. In addition, ECKENHOFF and JOHANSSON 1999 caution against extrapolating effects determined with clinically relevant anesthetic concentrations in vitro to in vivo systems, citing a lack of understanding of how various in vitro systems contribute to integrated responses in an intact organism. A clear advantage of using a genetic approach to investigate anesthetic action in vivo is that genes identified as altering cellular responses to these drugs must reflect molecular effects of anesthetics that are biologically relevant, although it does not demonstrate if this involvement is direct or indirect. In several model organisms, including yeast, concentrations higher than those used clinically have been employed to identify mutants with altered anesthetic response (OVERTON 1901 ; MORGAN et al. 1988 ; KEIL et al. 1996 ). One reason suggested for the necessity of high doses is the need of these organisms to protect themselves from potentially harmful compounds in an environment on which they exert little control (SEDENSKY et al. 1994 ). While this may be true, the findings reported here show that for yeast the relatively high biologically relevant anesthetic dose is due, at least in part, to: (1) the ability of yeast to efficiently import critical nutrients, (2) the culture conditions used to grow yeast in our lab, and (3) the particular auxotrophic markers present in our wild-type strain. Specifically, anesthetic MIC in yeast can be dramatically influenced by (1) the cellular concentration of a putative protein target [e.g., compare MIC for TAT1 (12%), YEpTAT1 (>14%), and tat1 (9%) strains (Fig 2 and Fig 3)], (2) the concentration of essential metabolites in the environment [e.g., compare MIC for the wild-type strain grown on 3x Leu 3x Trp (>14%) vs. 1x Leu 1x Trp (12%) vs. 0.5x Leu 0.5x Trp (9%; Fig 7 and Fig 9)], and (3) the auxotrophic requirements of the cell [e.g., compare MIC for the Leu^-Trp^- strain (12%) to that of the Leu⁺Trp⁺ strain (>14%; Fig 7A)]. Experiments are in progress to determine if strains auxotrophic for other nutrients have an even lower anesthetic MIC than the yeast strain currently used. The ability to manipulate MIC in yeast genetically or environmentally suggests that determination of effective concentrations in other in vivo or in vitro systems may be subject to similar constraints. Thus, finding that the effective concentration in a particular experimental system approximates a clinical concentration may be a fortuitous mix of experimental conditions and seems likely to provide limited insight regarding the biological importance of a given target.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

	ACKNOWLEDGMENTS

We thank Drs. Anita K. Hopper and Roderic G. Eckenhoff for their critical comments regarding this manuscript, members of the Keil laboratory for their helpful discussions, and M. Hall and M. C. Kielland-Brandt for providing plasmids used in this work. This work was supported in part by grant GM57822 from the National Institutes of Health to R.L.K.

Manuscript received January 28, 2002; Accepted for publication March 8, 2002.

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