Tryptophan recovers sensitivity to cell membrane stress in Saccharomyces cerevisiae

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Because deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate, we implemented a suppressor gene screening revealing that the TAT2 tryptophan permease rescues cell growth to sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that Δtrp1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as calcofluor white, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discover that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, our results expose a functional link between internal tryptophan levels and tryptophan biosynthesis in the response to plasma membrane damage.

suppressor gene screening revealing that the TAT2 tryptophan permease rescues cell growth to 23 sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of 24 tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that Δtrp1 25 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for 26 adenine, histidine, leucine or uracil. While also critical in the response to tea tree oil, TRP1 does 27 not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall 28 integrity signaling such as calcofluor white, Congo Red or heat stress. This implicates a distinction 29 Introduction 38 In the wild, yeast experience a variety of external conditions that cause stress, such as 39 changes in resource availability, temperature, osmotic fluctuations, oxidation, noxious chemicals, 40 pressure and physical stress. The yeast cell wall and plasma membrane are the first defensive 41 structures against external stress and are essential to acclimate to these conditions. In general, any 42 perturbation that disrupts the cell wall or membrane function activates a multifactorial stress 43 response in Saccharomyces cerevisiae (1,2). 44 Sodium Dodecyl Sulfate (SDS) is a common household detergent that permeates cell 45 membranes (3, 4), activates a stress response including Cell Wall Integrity (CWI) signaling and 46 restricts cell growth (5). The CWI pathway is a kinase cascade that responds to cell wall/membrane 47 perturbations in order to maintain cell integrity in yeast (1,2). Treatments that damage the yeast 48 cell wall or membrane such as chemicals like SDS (5), Calcofluor White (CFW) (6), Congo Red 49 (CR) (7) and Tea Tree Oil (TTO) (8) or by growth at elevated temperatures (9), trigger the CWI 50 pathway. 51 MCK1, the yeast homologue of the mammalian Glycogen Synthase Kinase-3 (GSK-3) (10, 52 11), is involved in a variety of stress response activities. Mck1p maintains genome integrity in 53 response to DNA damage (12,13) and is involved in the transcriptional regulation of stress 54 response genes (14,15). In addition, Mck1p is a downstream effector of CWI signaling activated 55 by high temperature, osmotic stress or calcium stress (16,17). Deletion of MCK1 causes hyper-56 sensitivity to SDS (14,16). We previously found that SDS induces cell cycle arrest during G1 57 phase via Mck1p (14). In order to understand the mechanism of cell growth inhibition by SDS, we 58 implemented a suppressor gene screening using Δmck1 cells in the presence of SDS. The screen 59 revealed that the TAT2 tryptophan permease rescued cell growth to SDS-treated Δmck1 cells. 60 The high affinity tryptophan permease, Tat2p (Tryptophan Amino acid Transporter), is a 61 constitutive permease regulated by the concentration of tryptophan in the media (18) itself exhibits protection from membrane disruptions. In addition to these cell wall/membrane 69 related stresses, it has been suggested that internal tryptophan levels influence growth recovery 70 post DNA damage (28, 29).

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Because our suppressor gene screening revealed TAT2 and that tryptophan is linked to 72 stress tolerance, we questioned the involvement of tryptophan in the recovery of cell growth in the 73 presence of SDS. In this work, we show that SDS-induced growth inhibition can be overcome with 74 exogenous tryptophan or tryptophan prototrophy. We found that tryptophan prototrophy exhibits 75 protection from growth inhibition due to particular cell wall/membrane damaging agents that 76 activate the CWI pathway, but not all treatments, suggesting that the need for tryptophan is 77 autonomous from CWI activity. In addition to tryptophan biosynthesis, we show that tyrosine 78 biosynthesis is also necessary for tolerance to SDS stress. Additionally, we determine that 79 tryptophan import is not disrupted by SDS exposure but enhanced. These results provide an 80 unknown connection to tryptophan and tyrosine in the protection from plasma membrane damage 81 that is not due to general nutrient starvation and is independent of CWI signaling. To affirm the rescue of Δmck1 sensitivity to SDS with TAT2, we cloned TAT2 into a pRS425 high 86 copy plasmid and asked if TAT2 alone rescues SDS-induced cell growth inhibition of Δmck1 cells.

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Indeed, we found that the TAT2 expressing plasmid conferred rescue to both Δmck1 and MCK1 88 cells in the presence of SDS (Fig 1A). It is known that during times of stress or nutrient starvation,

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Tat2p is sorted to the vacuole for degradation and then tryptophan uptake is maintained by Gap1p, 90 the General Amino acid Permease (30-32). This may explain why the SDS-induced growth 91 inhibition in Δmck1 cells was only mildly rescued by TAT2 overexpression.  To support the idea that cell growth sensitivity to SDS recovered by TAT2 overexpression is due 102 to tryptophan availability, we also observed that exogenous tryptophan recovered growth of both 103 Δmck1 and MCK1 cells when we supplemented YPD plates containing SDS with excess tryptophan 104 ( Fig 1B). However, cell growth was still inhibited by SDS with the addition of exogenous adenine, 105 histidine, leucine or uracil suggesting that recovery of SDS-induced growth inhibition is specific 106 to tryptophan. trp1-1, on SDS containing YPD plates. Indeed, the trp1-1 mutation itself caused sensitivity to SDS 118 in W303 cells (Fig 2A, section 5 and 6). From these results, we conclude that tryptophan 119 auxotrophy alone was sufficient to cause sensitivity to SDS, indicating the significance of TRP1 120 prototrophy for SDS resistance in multiple yeast backgrounds. These results prompted us to test further the prototrophic requirements for SDS resistance. W303 152 cells that are auxotrophic for all of the markers, ade2-1, his3-11,15, leu2-3,112, trp1-1 and ura3-153 1, show growth inhibition on YPD plus SDS plates (Fig 3, row 4). The matched cells harboring a 154 wild type copy of TRP1 and are auxotrophic for the other four genes, showed robust growth in the 155 presence of SDS whereas cells that were prototrophic for any other single mutant indicated besides 156 tryptophan show growth inhibition in the same conditions (Fig 3, rows 1, 5, 6, 7 and 8). As an 157 additional control, we used BY4741 cells that are prototrophic for TRP1 and as expected these 158 cells grow robustly at this concentration of SDS (Fig 3, row 2). These findings support the evidence 159 that trp1 auxotrophy has a more adverse effect to cells compromised with SDS than auxotrophy 160 for adenine, histidine, leucine or uracil. with either dye, heat stress or TTO triggers the CWI pathway (8,9,40,45). 190 We asked whether tryptophan prototrophy could recover growth sensitivity to cells challenged 191 with 10ug/ml CFW or 10ug/ml CR. In contrast to SDS treatment, we found that the different 192 varieties of W303 cells were as sensitive as each other upon CFW or CR treatment and this result 193 is regardless of tryptophan prototrophy (Fig 4B). We also found that BY4741 cells are not as 194 sensitive to CFW and CR as W303 cells (Fig 4B, row 7). 195 We also tested the effect of tryptophan prototrophy on cells compromised with heat stress. This 196 assay showed that both W303 and BY4741 cells grow well when incubated at 37ºC (Fig 4C, left).

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W303 cell growth was inhibited at 39ºC and like CFW and CR treatment; inhibition is completely 198 independent of tryptophan prototrophy (Fig 4C, right). BY4741 cells, however, do not show 199 growth sensitivity at 39ºC (Fig 4C, right, row 7).

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In converse to CFW, CR and heat stress, the presence of wild type TRP1 was able to recover cell 201 growth sensitivity due to TTO in prototrophic cells. W303 prototrophic cells were able to 202 overcome growth inhibition due to 0.15% TTO if they contain his3-11,15, but not trp1-1 (Fig 4D,   203 row 5 and 6). The W303 cells containing several auxotrophic markers could not grow in the 204 presence of 0.15% TTO, nor could the BY4741 cells indicating that multiple auxotrophies are also 205 detrimental for the TTO response (Fig 4D, rows 7 and 8). While the effects of TTO are not the 206 same as for SDS, they indicate that tryptophan prototrophy has a similar trend on growth recovery 207 to cells compromised with TTO as with SDS. These results indicate that the activity of the CWI 208 pathway is independent of tryptophan synthesis. Perhaps the stress response involving tryptophan 209 prototrophy is particular to membrane disruptions as opposed to cell wall perturbations.

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The TRP1 gene product is essential for yeast cells to biosynthesize tryptophan. S. cerevisiae uses 212 a shared pathway to synthesize tryptophan that also synthesizes phenylalanine and tyrosine (Fig   213   5A). We know that cells mutant for any of the enzymes specific to the tryptophan biosynthesis 214 pathway are sensitive to SDS (33). We wanted to know if either phenylalanine or tyrosine 215 auxotrophy causes the same response to SDS as an aberrant TRP1 gene.  This experiment uncovers that tyrosine is also required for yeast cells to survive SDS-treatment 246 along with a fully functional tryptophan biosynthesis pathway and provides additional evidence 247 that growth is influenced by tryptophan biosynthesis enzymes.

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It has been shown before that tryptophan can be imported through channels other than Tat2p, 249 primarily through Gap1p (18). We retrieved Δtat2 cells from the EUROSCARF deletion library 250 and compared their growth to Δtrp1 cells in the presence of SDS. In contrast to Δtrp1, Δtat2 cell 251 growth was not inhibited by SDS treatment (Fig 5C, row 3). We constructed a double deletion 252 mutant, Δtat2 Δtrp1, which would not be able to import tryptophan through Tat2p nor make its 253 own. Growth of the Δtat2 Δtrp1 double deletion mutant was mildly inhibited on SDS containing 254 plates compared to the single mutant, Δtat2. Yet, compared to the Δtrp1 single mutant, the 255 additional loss of Δtat2 conferred some growth resistance towards SDS treatment indicating that 256 indeed tryptophan uptake is maintained without Tat2p (Fig 5C, rows 3, 4 and 5). In this same 257 assay, we tested the growth of Δtat1 cells (Fig 5C, row 2). Tat1 encodes for the high affinity 258 tyrosine permease. We found that Δtat1 cell growth was not affected by SDS suggesting that 259 tyrosine uptake is also remediated by different means during an SDS response. These results 260 suggest that internal tryptophan and tyrosine levels are important during an SDS assault and that 261 they are acquired through uptake systems other than Tat2p and Tat1p. Because Δtrp1 cell growth 262 is so constrained by SDS suggest that utilization of the tryptophan biosynthesis pathway is also 263 significant during an SDS response. We considered that membrane disruptions caused by SDS could interrupt tryptophan uptake 268 systems and this is why TRP1 prototrophy is imperative to cells compromised with SDS. If SDS 269 inhibits amino acid import, our results indicate that it is specific for tryptophan and also for 270 tyrosine. To determine tryptophan uptake, we used prototrophic W303 cells whose growth is 271 uncompromised on YPD plates containing 0.0075% SDS (Fig 4A, row 4). We found that import 272 of radiolabeled L-[5-3H]tryptophan or L- [2,5-3H]histidine was enhanced within minutes upon 273 0.0075% SDS exposure compared to uncompromised cells in liquid culture (Fig 6). It is possible 274 that tryptophan and histidine leak into cells through membrane holes created by SDS at this 275 concentration and that is the explanation for enhanced uptake. However, we found the same 276 enhanced uptake when we challenged cells with a lower concentration of SDS at (0.005%. These 277 results suggest that cells are not starved for tryptophan or histidine upon SDS administration. It 278 has been shown that Gap1p activity can be produced within 5 minutes under certain conditions 279 (30-32). It is possible that Gap1p as a high capacity permease is activated by SDS treatment.

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Because tryptophan uptake is not inhibited by SDS treatment, provides further evidence that need 281 for tryptophan itself is important. Because we recovered the TAT2 tryptophan permease from a suppressor gene screen using Δmck1 289 cells in the presence of SDS, we studied tryptophan in the recovery from cell membrane stress due 290 to SDS exposure. First, we show that cells harboring Δtrp1 have a clear disadvantage in the 291 response to SDS compared to auxotrophies for adenine, histidine, leucine or uracil. Next, we found 292 that tryptophan prototrophy is also critical for stress tolerance towards TTO, another membrane 293 destabilizing drug. While both SDS and TTO cause CWI activation, we demonstrate that 294 tryptophan prototrophy is not able to alleviate growth inhibition due to other cell wall/membrane 295 damaging treatments that also activate the pathway indicating a distinction from CWI signaling.

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This also implicates that the resistance to growth inhibition shown by tryptophan prototrophic cells 297 may be specific to the type of membrane damage created by SDS and TTO as opposed to cell wall 298 disruptions. In addition, we uncover that tyrosine biosynthesis is also important for resistance to 299 SDS-induced growth inhibition whereas phenylalanine biosynthesis is dispensable. We also found 300 that in the presence of SDS, Δtat2 deletion cells show increased growth resistance to Δtrp1 cells 301 indicating that internal tryptophan levels are maintained during an SDS assault through uptake 302 systems other than Tat2p. Finally, we observe that both tryptophan and histidine import becomes 303 enhanced immediately upon addition of SDS as a further indication that SDS-induced growth 304 inhibition is not due to nutrient starvation in general.

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These results suggest that tryptophan, tryptophan biosynthesis and tyrosine biosynthesis play a 306 role in the plasma membrane stress response. It is thought that the constitutive permeases, such as 307 Tat2p and Tat1p, uptake amino acids for use in protein synthesis. Gap1p, however, is a transporter 308 of all amino acids and is regulated by nitrogen (47) therefore it is thought that Gap1p acquires 309 amino acids for use as a nitrogen source (48). Future directions would be to explore tryptophan The yeast cells used are derivatives of W303 (strain list in Table 1) except for the BY4741 control  The suppressor gene screen, which has been described in (49) Amino acid uptake assay 362 The protocol was adapted from J Heitman (51). LSY119 cells in log-phase were harvested and 363 washed once with 10mM sodium citrate, pH4.5, and resuspended in 50mLs of 10mM sodium 364 citrate, pH4.5, containing 20mM ammonium sulfate and 2% glucose. SDS at 0.0075% was added 365 and the 0 time point was taken immediately before the radioactive substrate addition. Uptake was 366 assayed by adding 0.5mL of radiolabeled amino acid mixture to 4.5mL cell culture. The