Structure of the Yeast Cell Wall Integrity Sensor Wsc1 Reveals an Essential Role of Surface-Exposed Aromatic Clusters
Abstract
:1. Introduction
2. Materials and Methods
2.1. Yeast Strains and Growth Tests
2.2. Plasmids
2.3. Recombinant Production of CRD Proteins
2.4. CD Spectroscopy and Thermal Shift Assays
2.5. Crystallization
2.6. X-ray Data Collection, Structure Solution, and Analysis
2.7. Western-Blot Analysis
2.8. Microscopy
2.9. Bioinformatic Analysis
2.10. Data Availability
3. Results
3.1. Characterization of Recombinant CRDs from S. cerevisiae Wsc1, Wsc2, and Wsc3
3.2. High-Resolution Structure of the ScWsc1 CRD
3.3. Functional Characterization of Wsc1 Surface-Exposed Aromatic Clusters
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Garcia-Rubio, R.; de Oliveira, H.C.; Rivera, J.; Trevijano-Contador, N. The Fungal Cell Wall: Candida, Cryptococcus, and Aspergillus Species. Front. Microbiol. 2019, 10, 2993. [Google Scholar] [CrossRef] [PubMed]
- Gow, N.A.R.; Latge, J.P.; Munro, C.A. The Fungal Cell Wall: Structure, Biosynthesis, and Function. Microbiol. Spectr. 2017, 5. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- De Groot, P.W.; Hellingwerf, K.J.; Klis, F.M. Genome-wide identification of fungal GPI proteins. Yeast 2003, 20, 781–796. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lesage, G.; Bussey, H. Cell wall assembly in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2006, 70, 317–343. [Google Scholar] [CrossRef] [Green Version]
- Orlean, P. Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall. Genetics 2012, 192, 775–818. [Google Scholar] [CrossRef] [Green Version]
- Levin, D.E. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2005, 69, 262–291. [Google Scholar] [CrossRef] [Green Version]
- Elorza, M.V.; Rico, H.; Sentandreu, R. Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. J. Gen. Microbiol. 1983, 129, 1577–1582. [Google Scholar] [CrossRef] [Green Version]
- Roncero, C.; Duran, A. Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: In vivo activation of chitin polymerization. J. Bacteriol. 1985, 163, 1180–1185. [Google Scholar] [CrossRef] [Green Version]
- Ketela, T.; Green, R.; Bussey, H. Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J. Bacteriol. 1999, 181, 3330–3340. [Google Scholar] [CrossRef] [Green Version]
- Garcia, R.; Bermejo, C.; Grau, C.; Perez, R.; Rodriguez-Pena, J.M.; Francois, J.; Nombela, C.; Arroyo, J. The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J. Biol. Chem. 2004, 279, 15183–15195. [Google Scholar] [CrossRef] [Green Version]
- Imai, K.; Noda, Y.; Adachi, H.; Yoda, K. A novel endoplasmic reticulum membrane protein Rcr1 regulates chitin deposition in the cell wall of Saccharomyces cerevisiae. J. Biol. Chem. 2005, 280, 8275–8284. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bermejo, C.; Garcia, R.; Straede, A.; Rodriguez-Pena, J.M.; Nombela, C.; Heinisch, J.J.; Arroyo, J. Characterization of sensor-specific stress response by transcriptional profiling of wsc1 and mid2 deletion strains and chimeric sensors in Saccharomyces cerevisiae. Omics J. Integr. Biol. 2010, 14, 679–688. [Google Scholar] [CrossRef] [PubMed]
- Rodicio, R.; Heinisch, J.J. Together we are strong—Cell wall integrity sensors in yeasts. Yeast 2010, 27, 531–540. [Google Scholar] [CrossRef]
- Dichtl, K.; Samantaray, S.; Wagener, J. Cell wall integrity signalling in human pathogenic fungi. Cell Microbiol. 2016, 18, 1228–1238. [Google Scholar] [CrossRef] [PubMed]
- Levin, D.E. Regulation of cell wall biogenesis in Saccharomyces cerevisiae: The cell wall integrity signaling pathway. Genetics 2011, 189, 1145–1175. [Google Scholar] [CrossRef] [Green Version]
- Heinisch, J.J.; Rodicio, R. Protein kinase C in fungi-more than just cell wall integrity. FEMS Microbiol. Rev. 2018, 42, fux051. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kock, C.; Dufrene, Y.F.; Heinisch, J.J. Up against the wall: Is yeast cell wall integrity ensured by mechanosensing in plasma membrane microdomains? Appl. Environ. Microbiol. 2015, 81, 806–811. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jacoby, J.J.; Nilius, S.M.; Heinisch, J.J. A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene. Mol. Gen. Genet. 1998, 258, 148–155. [Google Scholar] [CrossRef]
- Lodder, A.L.; Lee, T.K.; Ballester, R. Characterization of the Wsc1 protein, a putative receptor in the stress response of Saccharomyces cerevisiae. Genetics 1999, 152, 1487–1499. [Google Scholar] [CrossRef]
- Heinisch, J.J.; Dupres, V.; Wilk, S.; Jendretzki, A.; Dufrene, Y.F. Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1. PLoS ONE 2010, 5, e11104. [Google Scholar] [CrossRef] [Green Version]
- Dupres, V.; Alsteens, D.; Wilk, S.; Hansen, B.; Heinisch, J.J.; Dufrene, Y.F. The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo. Nat. Chem. Biol. 2009, 5, 857–862. [Google Scholar] [CrossRef] [PubMed]
- Kock, C.; Arlt, H.; Ungermann, C.; Heinisch, J.J. Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling. Cell Microbiol. 2016, 18, 1251–1267. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Voskoboynikova, N.; Karlova, M.; Kurre, R.; Mulkidjanian, A.Y.; Shaitan, K.V.; Sokolova, O.S.; Steinhoff, H.J.; Heinisch, J.J. A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy. J. Fungi 2021, 7, 118. [Google Scholar] [CrossRef] [PubMed]
- Rodicio, R.; Buchwald, U.; Schmitz, H.P.; Heinisch, J.J. Dissecting sensor functions in cell wall integrity signaling in Kluyveromyces lactis. Fungal Genet. Biol. 2008, 45, 422–435. [Google Scholar] [CrossRef] [PubMed]
- Futagami, T.; Nakao, S.; Kido, Y.; Oka, T.; Kajiwara, Y.; Takashita, H.; Omori, T.; Furukawa, K.; Goto, M. Putative stress sensors WscA and WscB are involved in hypo-osmotic and acidic pH stress tolerance in Aspergillus nidulans. Eukaryot. Cell 2011, 10, 1504–1515. [Google Scholar] [CrossRef] [Green Version]
- Maddi, A.; Dettman, A.; Fu, C.; Seiler, S.; Free, S.J. WSC-1 and HAM-7 are MAK-1 MAP kinase pathway sensors required for cell wall integrity and hyphal fusion in Neurospora crassa. PLoS ONE 2012, 7, e42374. [Google Scholar] [CrossRef] [Green Version]
- Tong, S.M.; Chen, Y.; Zhu, J.; Ying, S.H.; Feng, M.G. Subcellular localization of five singular WSC domain-containing proteins and their roles in Beauveria bassiana responses to stress cues and metal ions. Environ. Microbiol. Rep. 2016, 8, 295–304. [Google Scholar] [CrossRef]
- Cohen-Kupiec, R.; Broglie, K.E.; Friesem, D.; Broglie, R.M.; Chet, I. Molecular characterization of a novel beta-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum. Gene 1999, 226, 147–154. [Google Scholar] [CrossRef]
- Wawra, S.; Fesel, P.; Widmer, H.; Neumann, U.; Lahrmann, U.; Becker, S.; Hehemann, J.H.; Langen, G.; Zuccaro, A. FGB1 and WSC3 are in planta-induced beta-glucan-binding fungal lectins with different functions. New Phytol. 2019, 222, 1493–1506. [Google Scholar] [CrossRef] [Green Version]
- Mao, B.; Wu, W.; Davidson, G.; Marhold, J.; Li, M.; Mechler, B.M.; Delius, H.; Hoppe, D.; Stannek, P.; Walter, C.; et al. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 2002, 417, 664–667. [Google Scholar] [CrossRef]
- Nakamura, T.; Nakamura, T.; Matsumoto, K. The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology. J. Cell Mol. Med. 2008, 12, 391–408. [Google Scholar] [CrossRef] [PubMed]
- Zebisch, M.; Jackson, V.A.; Zhao, Y.; Jones, E.Y. Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf. Structure 2016, 24, 1599–1605. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pereira, G.; Tanaka, T.U.; Nasmyth, K.; Schiebel, E. Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. EMBO J. 2001, 20, 6359–6370. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Janke, C.; Magiera, M.M.; Rathfelder, N.; Taxis, C.; Reber, S.; Maekawa, H.; Moreno-Borchart, A.; Doenges, G.; Schwob, E.; Schiebel, E.; et al. A versatile toolbox for PCR-based tagging of yeast genes: New fluorescent proteins, more markers and promoter substitution cassettes. Yeast 2004, 21, 947–962. [Google Scholar] [CrossRef] [PubMed]
- Guthrie, C.; Fink, G.R. (Eds.) Guide to Yeast Genetics and Molecular Biology; Academic Press: San Diego, CA, USA, 1991; Volume 194, pp. 1–863. [Google Scholar]
- Lutz, A.P.; Schladebeck, S.; Renicke, C.; Spadaccini, R.; Mosch, H.U.; Taxis, C. Proteasome Activity Is Influenced by the HECT_2 Protein Ipa1 in Budding Yeast. Genetics 2018, 209, 157–171. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sikorski, R.S.; Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989, 122, 19–27. [Google Scholar] [CrossRef]
- Veelders, M.; Brückner, S.; Ott, D.; Unverzagt, C.; Mösch, H.U.; Essen, L.O. Structural basis of flocculin-mediated social behavior in yeast. Proc. Natl. Acad. Sci. USA 2010, 107, 22511–22516. [Google Scholar] [CrossRef] [Green Version]
- Kabsch, W. Xds. Acta Crystallogr. 2010, 66, 125–132. [Google Scholar] [CrossRef] [Green Version]
- Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. 2010, 66, 133–144. [Google Scholar] [CrossRef] [Green Version]
- Evans, P.R. An introduction to data reduction: Space-group determination, scaling and intensity statistics. Acta Crystallogr. 2011, 67, 282–292. [Google Scholar] [CrossRef] [Green Version]
- Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.; McCoy, A.; et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. 2011, 67, 235–242. [Google Scholar] [CrossRef] [Green Version]
- McCoy, A.J.; Grosse-Kunstleve, R.W.; Adams, P.D.; Winn, M.D.; Storoni, L.C.; Read, R.J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658–674. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Adams, P.D.; Afonine, P.V.; Bunkoczi, G.; Chen, V.B.; Davis, I.W.; Echols, N.; Headd, J.J.; Hung, L.W.; Kapral, G.J.; Grosse-Kunstleve, R.W.; et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. 2010, 66, 213–221. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and development of Coot. Acta Crystallogr. 2010, 66, 486–501. [Google Scholar] [CrossRef] [Green Version]
- Yaffe, M.P.; Schatz, G. Two nuclear mutations that block mitochondrial protein import in yeast. Proc. Natl. Acad. Sci. USA 1984, 81, 4819–4823. [Google Scholar] [CrossRef] [Green Version]
- Madeira, F.; Park, Y.M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.R.N.; Potter, S.C.; Finn, R.D.; et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019, 47, W636–W641. [Google Scholar] [CrossRef] [Green Version]
- Dupres, V.; Heinisch, J.J.; Dufrene, Y.F. Atomic force microscopy demonstrates that disulfide bridges are required for clustering of the yeast cell wall integrity sensor Wsc1. Langmuir 2011, 27, 15129–15134. [Google Scholar] [CrossRef]
- Ultsch, M.; Lokker, N.A.; Godowski, P.J.; de Vos, A.M. Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 A resolution. Structure 1998, 6, 1383–1393. [Google Scholar] [CrossRef] [Green Version]
- Wong, S.S.; Ostergaard, S.; Hall, G.; Li, C.; Williams, P.M.; Stennicke, H.; Emsley, J. A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain. Blood 2016, 127, 2915–2923. [Google Scholar] [CrossRef] [Green Version]
- Mathieu, Y.; Offen, W.A.; Forget, S.M.; Ciano, L.; Viborg, A.H.; Blagova, E.; Henrissat, B.; Walton, P.H.; Davies, G.J.; Brumer, H. Discovery of a Fungal Copper Radical Oxidase with High Catalytic Efficiency toward 5-Hydroxymethylfurfural and Benzyl Alcohols for Bioprocessing. ACS Catal. 2020, 10, 3042–3058. [Google Scholar] [CrossRef]
- Crooks, G.E.; Hon, G.; Chandonia, J.M.; Brenner, S.E. WebLogo: A sequence logo generator. Genome Res. 2004, 14, 1188–1190. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ovchinnikov, S.; Kamisetty, H.; Baker, D. Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information. eLife 2014, 3, e02030. [Google Scholar] [CrossRef] [PubMed]
- Kraushaar, T.; Brückner, S.; Veelders, M.; Rhinow, D.; Schreiner, F.; Birke, R.; Pagenstecher, A.; Mösch, H.-U.; Essen, L.-O. Interactions by the Fungal Flo11 Adhesin Depend on a Fibronectin Type III-like Adhesin Domain Girdled by Aromatic Bands. Structure 2015, 23, 1005–1017. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reithofer, V.; Fernandez-Pereira, J.; Alvarado, M.; de Groot, P.; Essen, L.O. A novel class of Candida glabrata cell wall proteins with beta-helix fold mediates adhesion in clinical isolates. PLoS Pathog. 2021, 17, e1009980. [Google Scholar] [CrossRef] [PubMed]
- Lanzarotti, E.; Biekofsky, R.R.; Estrin, D.A.; Marti, M.A.; Turjanski, A.G. Aromatic-aromatic interactions in proteins: Beyond the dimer. J. Chem. Inf. Model 2011, 51, 1623–1633. [Google Scholar] [CrossRef] [PubMed]
- Lanzarotti, E.; Defelipe, L.A.; Marti, M.A.; Turjanski, A.G. Aromatic clusters in protein-protein and protein-drug complexes. J. Cheminform. 2020, 12, 30. [Google Scholar] [CrossRef] [PubMed]
- Brückner, S.; Schubert, R.; Kraushaar, T.; Hartmann, R.; Hoffmann, D.; Jelli, E.; Drescher, K.; Müller, D.J.; Oliver Essen, L.O.; Mösch, H.U. Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family. eLife 2020, 9, e55587. [Google Scholar] [CrossRef]
- Maestre-Reyna, M.; Diderrich, R.; Veelders, M.S.; Eulenburg, G.; Kalugin, V.; Brückner, S.; Keller, P.; Rupp, S.; Mösch, H.U.; Essen, L.O. Structural basis for promiscuity and specificity during Candida glabrata invasion of host epithelia. Proc. Natl. Acad. Sci. USA 2012, 109, 16864–16869. [Google Scholar] [CrossRef] [Green Version]
- Diderrich, R.; Kock, M.; Maestre-Reyna, M.; Keller, P.; Steuber, H.; Rupp, S.; Essen, L.O.; Mösch, H.U. Structural Hot Spots Determine Functional Diversity of the Candida glabrata Epithelial Adhesin Family. J. Biol. Chem. 2015, 290, 19597–19613. [Google Scholar] [CrossRef] [Green Version]
- Kock, M.; Brückner, S.; Wozniak, N.; Maestre-Reyna, M.; Veelders, M.; Schlereth, J.; Mösch, H.U.; Essen, L.O. Structural and Functional Characterization of PA14/Flo5-Like Adhesins From Komagataella pastoris. Front. Microbiol. 2018, 9, 2581. [Google Scholar] [CrossRef] [Green Version]
- Hoffmann, D.; Diderrich, R.; Reithofer, V.; Friederichs, S.; Kock, M.; Essen, L.O.; Mösch, H.U. Functional reprogramming of Candida glabrata epithelial adhesins: The role of conserved and variable structural motifs in ligand binding. J. Biol. Chem. 2020, 295, 12512–12524. [Google Scholar] [CrossRef] [PubMed]
- Essen, L.O.; Vogt, M.S.; Mösch, H.U. Diversity of GPI-anchored fungal adhesins. Biol. Chem. 2020, 401, 1389–1405. [Google Scholar] [CrossRef] [PubMed]
Plasmid | Relevant Genotype | Source |
---|---|---|
pET-28(a)+ | PT7-6xHis lacI KanR | Merck, Germany |
BHUM3120 | WSC1(22−118) in pET-28(a)+ | this study |
BHUM3121 | WSC2(24−118) in pET-28(a)+ | this study |
BHUM3122 | WSC3(40−133) in pET-28(a)+ | this study |
pFA6a-natNT2 | PFA6a-NatNT2 AmpR | [34] |
pCR95 | mNeonGreen::NatNT2 AmpR | [36] |
pRS314 | TRP1 ARS CEN2 AmpR | [37] |
BHUM3291 | WSC1 in pRS314 | this study |
BHUM3293 | WSC1Y22A Y24A Y107A in pRS314 | this study |
BHUM3295 | WSC1Y64A Y70A Y104A in pRS314 | this study |
BHUM3297 | WSC1Y41A W43A Y89A F91A Y93A in pRS314 | this study |
BHUM3301 | WSC1∆CRD in pRS314 | this study |
BHUM3303 | WSC1-mNeonGreen in pRS314 | this study |
BHUM3304 | WSC1Y22A Y24A Y107A-mNeonGreen in pRS314 | this study |
BHUM3305 | WSC1Y64A Y70A Y104A-mNeonGreen in pRS314 | this study |
BHUM3306 | WSC1Y41A W43A Y89A F91A Y93A-mNeonGreen in pRS314 | this study |
BHUM3308 | WSC1∆CRD-mNeonGreen in pRS314 | this study |
PDB Code | 7PZ2 |
---|---|
X-ray source | ESRF ID29 |
Wavelength (Å) | 0.979 |
Resolution range (Å) 1 | 26.75–1.58 (1.64–1.58) |
Space group | P 1 21 1 |
Unit cell | a = 31.82 Å, b = 53.5 Å, c = 51.93 Å α = 90°, β = 95.46°, γ = 90° |
Total reflections 1 | 70,058 (6136) |
Unique reflections 1 | 23,125 (2133) |
Multiplicity 1 | 3.0 (2.8) |
Completeness (%) 1 | 97.06 (91.42) |
Mean I/sigma(I) 1 | 10.35 (2.17) |
Wilson B-factor (Å2) | 16.72 |
Rmerge 1 | 0.06339 (0.4653) |
CC1/2 1 | 0.997 (0.756) |
CC* 1 | 0.999 (0.928) |
Reflections used in refinement 1 | 23,052 (2132) |
Reflections used for Rfree 1 | 1113 (126) |
Rwork 1 | 0.150 (0.258) |
Rfree 1 | 0.184 (0.251) |
CC (work, free) 1 | 0.973 (0.819), 0.965 (0.824) |
Number of non-hydrogen atoms | 1731 |
Macromolecules | 1527 |
Ligands, solvent | 7197 |
Protein residues | 197 |
r.m.s.d. bonds (Å) | 0.005 |
r.m.s.d. angles (°) | 0.99 |
Ramachandran favored (%) | 92.23 |
Ramachandran allowed (%) | 7.77 |
Ramachandran outliers (%) | 0.00 |
Rotamer outliers (%) | 0.56 |
Clashscore 2 | 1.06 |
Average B-factor (Å2) | 20.88 |
Macromolecules (Å2) | 19.53 |
Ligands, Solvent (Å2) | 25.84, 31.15 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Schöppner, P.; Lutz, A.P.; Lutterbach, B.J.; Brückner, S.; Essen, L.-O.; Mösch, H.-U. Structure of the Yeast Cell Wall Integrity Sensor Wsc1 Reveals an Essential Role of Surface-Exposed Aromatic Clusters. J. Fungi 2022, 8, 379. https://doi.org/10.3390/jof8040379
Schöppner P, Lutz AP, Lutterbach BJ, Brückner S, Essen L-O, Mösch H-U. Structure of the Yeast Cell Wall Integrity Sensor Wsc1 Reveals an Essential Role of Surface-Exposed Aromatic Clusters. Journal of Fungi. 2022; 8(4):379. https://doi.org/10.3390/jof8040379
Chicago/Turabian StyleSchöppner, Philipp, Anne Pia Lutz, Bernard Johannes Lutterbach, Stefan Brückner, Lars-Oliver Essen, and Hans-Ulrich Mösch. 2022. "Structure of the Yeast Cell Wall Integrity Sensor Wsc1 Reveals an Essential Role of Surface-Exposed Aromatic Clusters" Journal of Fungi 8, no. 4: 379. https://doi.org/10.3390/jof8040379