The FASEB Journal • Research Communication Stress-driven structural and functional switching of Ypt1p from a GTPase to a molecular chaperone mediates thermo tolerance in Saccharomyces cerevisiae Chang Ho Kang,* ,1 Sun Yong Lee,* ,1 Joung Hun Park,* Yuno Lee,* Hyun Suk Jung, † Yong Hun Chi,* Young Jun Jung,* Ho Byoung Chae,* Mi Rim Shin,* Woe Yeon Kim,* Dae-Jin Yun,* and Sang Yeol Lee* ,2 *Division of Applied Life Science and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea; † Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon, South Korea ABSTRACT Guanosine triphosphatases (GTPases) function as molecular switches in signal transduction pathways that enable cells to respond to extracellular stimuli. Saccharomyces cerevisiae yeast protein two 1 protein (Ypt1p) is a monomeric small GTPase that is essential for endoplasmic reticulum-to-Golgi trafficking. By size- exclusion chromatography, SDS-PAGE, and native PAGE, followed by immunoblot analysis with an anti-Ypt1p anti- body, we found that Ypt1p structurally changed from low- molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes after heat shock. Based on our results, Ypt1p exhibited dual functions both as a GTPase and a molecular chaperone, and furthermore, heat shock in- duced a functional switch from that of a GTPase to a mo- lecular chaperone driven by the structural change from LMW to HMW forms. Subsequently, we found, by using a galactose-inducible expression system, that conditional overexpression of YPT1 in yeast cells enhanced the ther- motolerance of cells by increasing the survival rate at 55°C by ∼60%, compared with the control cells expressing YPT1 in the wild-type level. Altogether, our results suggest that Ypt1p is involved in the cellular protection process under heat stress conditions. Also, these findings provide new insight into the in vivo roles of small GTP-binding proteins and have an impact on research and the investigation of human diseases.—Kang, C. H., Lee, S. Y., Park, J. H., Lee, Y., Jung, H. S., Chi, Y. H., Jung, Y. J., Chae, H. B., Shin, M.R., Kim, W. Y., Yun, D.-J., Lee, S. Y. Stress-driven structural and functional switching of Ypt1p from a GTPase to a molecular chaperone mediates thermo tolerance in Saccharomyces cerevisiae. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: heat shock • small GTPase • structural change Organisms constantly have to adjust to their environment because of varying nutrient availability, osmotic imbal- ances, fluctuating temperatures, and hazardous mole- cules. Chemical or physical stresses induce denaturation, loss of activity, and mislocalization of cellular proteins. In addition, high temperature negatively affects membrane- linked processes by altering membrane fluidity and per- meability (1–3). Several stresses, including heat shock, in- crease generation of reactive oxygen species (ROS) in cells, resulting in several detrimental effects, including pro- grammed cell death (4, 5). Cells adjust and survive in the stressful environment by the speci fic reprogramming of gene expression. For example, organisms can grow at tem- peratures above their optimum by activating a protective transcriptional program termed the heat-shock response and by altering their defense signaling pathways to change the expression level of defense genes, carbohydrate flux, and membrane compositions (6, 7). To protect themselves from various external stresses and stress-mediated protein unfolding and aggregation (8, 9), all aerobic organisms are equipped with a wide range of antioxidant proteins and diverse molecular chaperones (10, 11). Recently, it has been shown that several proteins are involved in cellular protection against heat stress by virtue of their ability to acquire new functions through heat- induced structural changes (12–15). For example, 2 cyto- solic peroxiredoxins (Prxs) of yeast, cPrxI and -II, have been shown to switch from low-molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes in response to heat shock and radical stresses (16). The conformational change is accompanied by functional switching from a peroxidase to a molecular chaperone. Consequently, null cPrxI and -II mutations in yeast result in hypersensitivity to heat-shock stress. On the basis of these observations, we screened for protein extracts of Abbreviations: bis-ANS, 4,49-dianilino-1,19-binaphthyl-5,59- disulfonic acid, dipotassium salt; CBB, Coomassie Brilliant Blue; CS, citrate synthase; cPrxI, cytosolic peroxiredoxin I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; GTPase, guanosine triphosphatase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ( continued on next page) 1 These authors contributed equally to this work. 2 Correspondence: Division of Applied Life Science and PMBBRC, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, South Korea. E-mail: sylee@gnu.ac.kr doi: 10.1096/fj.15-270140 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 0892-6638/15/0029-0001 © FASEB 1 The FASEB Journal article fj.15-270140. Published online July 13, 2015. 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