Benzyl Alcohol Increases Diffusion Limit of Nuclear Membrane in Saccharomyces cerevisiae Cells

Year 2022, , 26 - 30, 30.06.2022

Bengü Ergüden

https://doi.org/10.26650/EurJBiol.2022.1058174

Abstract

Objective: Fungi are invasive species responsible for infections in many people around the world and which severely affect the immune system. The opportunistic pathogenic species, such as Candida species and Aspergillus fumigatus, can cause death in people with weakened immune systems. Natural medicines derived from plants are often used to treat fungal diseases. In connection with our efforts to unearth possible cellular targets of antimicrobial agents, in this study, we aimed to determine the functional consequences of benzyl alcohol treatment on the nuclear membrane. Materials and Methods: We analysed the nuclear membrane distortions caused by benzyl alcohol in Saccharomyces cerevisiae cells using Nup49-GFP reporter strain. We also studied cellular distributions of various fluorescently tagged nuclearcytoplasmic shuttling proteins to determine any functional disturbances in nuclear pore complexes upon benzyl alcohol treatment. Localization of 51.5 kDa protein LexA-NES-GFP and 61.8 kDa protein Pho4(Δ157-164)-GFP to the nucleus in yeast cells was key for evaluating the effect upon diffusion limit of pores. Results: By analyzing the distribution of fluorescently tagged nuclear localization signal or nuclear export signals bearing reporter proteins between the nucleus and cytoplasm, we have shown that the nuclear membrane becomes leaky upon benzyl alcohol treatment. Conclusion: The diffusion limit across the nuclear membrane in yeast cells is increased upon benzyl alcohol treatment. We believe that these findings not only increase our understanding of the mode of action of benzyl alcohol bearing antifungal agents, but also help widening their use.

Keywords

Benzyl alcohol, yeast, antifungal activity, nuclear membrane, nuclear pore complex

References

• 1. D’Angelo MA and Hetzer MW. The role of the nuclear envelope in cellular organization. Cell Mol Life Sci 2006; 63: 316-32.
• 2. Cook A, Bono F, Jinek M, Conti E. Structural biology of nucleocytoplasmic transport. Annu Rev Biochem 2007; 76: 647-71.
• 3. Yang Q, Rout MP, Akey CW. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol Cell 1998; 1: 223-34.
• 4. Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol 2000; 148: 635-51.
• 5. Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol 2002; 158: 915-27.
• 6. Reichelt R, Holzenburg A, Buhle EL Jr, Jarnik M, Engel A, Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol 1990; 110: 883-94.
• 7. Mosammaparast N, Pemberton LF. Karyopherins: from nuclear- transport mediators to nuclear-function regulators. Trends Cell Biol 2004; 14: 547-56.
• 8. Mansfeld J, Guttinger S, Hawryluk-Gara LA, Pante N, Mall M, Galy V, Haselmann U, Muhlhausser P, Wozniak RW, Mattaj IW, Kutay U, Antonin W. The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells. Mol Cell 2006; 22: 93-103.
• 9. Stavru F, Hulsmann BB, Spang A, Hartmann E, Cordes VC, Görlich D. NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes. J Cell Biol 2006; 173: 509-19.
• 10. Schwartz TU. Modularity within the architecture of the nuclear pore complex. Curr Opin Struct Biol 2005; 15: 221-6.
• 11. Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, Suprapto A, Karni-Schmidt O, Williams R, Chait BT, Rout MP, Sali A. Determining the architectures of macromolecular assemblies. Nature 2007; 450: 683-94.
• 12. Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, Suprapto A, Karni-Schmidt O, Williams R, Chait BT, Sali A, Rout MP. The molecular architecture of the nuclear pore complex. Nature 2007; 450: 695-701.
• 13. Suntharalingam M, Wente SR. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev Cell 2003; 4: 775-89.
• 14. Macara IG. Transport into and out of the nucleus. Microbiol Mol Biol Rev 2001; 65: 570-94.
• 15. Konuk HB, Ergüden B. Phenolic –OH group is crucial for the antifungal activity of terpenoids via disruption of cell membrane integrity. Folia Microbiololica 2020; 65: 775-83.
• 16. Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989; 122: 9-27.
• 17. Ergüden B. Saccharomyces cerevisiae Outer and Inner Membranes are Compromised upon Benzyl Alcohol Treatment. Int J Memb Sci Tech 2021; 8: 35-9.
• 18. Thaller DJ, Lusk CP. Fantastic nuclear envelope herniations and where to find them. Biochem Soc Trans 2018; 46: 877-89.
• 19. Sezen B. Reduction of Saccharomyces cerevisiae Pom34 protein level by SESA network is related to membrane lipid composition. FEMS Yeast Research 2015; 15: fov089.
• 20. Madrid AS, Mancuso J, Cande WZ, Weis K. The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function. J Cell Biol 2006; 173: 361-71.
• 21. Meseroll RA, Cohen-Fix O. The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations. J Cell Physiol 2016; 231: 2353-60.
• 22. Brown JT, Alexandra JH, Christopher MW, Belanger KD. Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae. Biology Open 2021; 10: bio057661.
• 23. Gilbert W, Siebel CW, Guthrie C. Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation. RNA 2001; 7: 302- 13.
• 24. Sanchez NS, Königsberg M. Using Yeast to Easily Determine Mitochondrial Functionality with 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyltetrazolium Bromide (MTT) Assay. Biochem Mol Biol Edu 2006; 34: 209-12.
• 25. Frey S, Richter RP, Gorlich D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 2006; 314: 815-17.
• 26. Ribbeck K, Gorlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J 2002; 21: 2664-71.
• 27. Ribbeck K, Gorlich D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J 2001; 20: 1320–30.
• 28. Takahashi U, Hamada K, Iwahashi H. Critical Damage to the Cellular Organelles of Saccharomyces cerevisiae Under Sublethal Conditions Upon High Pressure Carbon Dioxide Treatment. High Pressure Res 2019; 39: 273-9.