The Effect of Tec1 Transcription Factor on NTH1 Gene Expression

Yıl 2019, Cilt: 12 Sayı: 2, 620 - 627, 31.08.2019

Tulay Turgut Genc Selen Çakas

https://doi.org/10.18185/erzifbed.457862

Öz

The intracellular accumulation of trehalose is one of the physiological response of yeast cells to different environmental stresses. The amount of trehalose in the cell changes during the phases of cell cycle and affects the chronological life span of Saccharomyces cerevisiae yeast cells. The expression of the NTH1 gene, which encodes the neutral trehalase enzyme responsible from the stress-accumulated trehalose breakdown, is controlled through different signaling pathways. TOR signaling system, one of these pathways, regulates the stability of Tec1 protein which controls the chronological life span of yeast cells. The detection of TCS elements in NTH1 promotor region enhances the probability of NTH1 gene regulation by Tec1. In order to determine the effect of Tec1 transcription factor on NTH1 gene expression, Nth1-LacZ gene fusion plasmid was transformed to wild type and Δtec1 mutant S. cerevisiae yeast cells. The amount of NTH1 gene expression and trehalose accumulation were determined in the presence of good and poor nitrogen sources both in wild type and mutant yeast cells. The results showed that in the absence of Tec1 protein NTH1 gene was activated and trehalose accumulation decreased considerably at normal growth conditions. These results revealed that, Tec1 transcription factor is essential for negative regulation of NTH1 transcription, especially under abundance of nutrient.

Anahtar Kelimeler

NTH1, Tec1p, trehalose, Saccharomyces cerevisiae

Tec1 Transkripsiyon Faktörünün NTH1 Gen Ekspresyonuna Etkisi

Öz

Hücre
içi trehaloz birikimi maya hücrelerinin farklı çevresel streslere karşı
oluşturduğu fizyolojik cevaplardan birisidir. Saccharomyces cerevisiae maya hücrelerinde trehaloz miktarı hücre
döngüsünün farklı aşamalarında farklı miktarlarda bulunmakta olup kronolojik
yaşam uzunluğunu etkilemektedir. Stres koşullarında biriktirilen trehalozun
yıkımından sorumlu nötral trehalaz enzimini kodlayan NTH1 geninin ekspresyonu farklı sinyal yolakları üzerinden kontrol
edilmektedir. Bu yolaklardan bir tanesi TOR sinyal yolağı olup aynı zamanda bu
yolak maya hücrelerinin kronolojik yaşam uzunluğunu etkileyen Tec1
transkripsiyon faktörünün de stabilitesini etkilemektedir. Trehaloz sentezinden
ve yıkımından sorumlu genlerin promotor bölgelerinin analizinde NTH1 promotorundan tespit edilen TCS
elementleri Tec1 proteinin NTH1
geninin regülasyonunda görev alma olsılığını kuvvetlendirmektedir. Tec1
transkripsiyon faktörünün NTH1 gen
ekspresyonuna etkisini belirlemek amacıyla, yaban tip ve ∆tec1 S. cerevisiae maya
hücrelerine NTH1 promotor bölgesi ve LacZ gen füzyonu içeren
plazmit aktarılmıştır. Kuvvetli azot ve zayıf azot kaynaklarında, Tec1
proteinin varlığında ve yokluğunda maya hücrelerinin trahaloz birikimleri ve NTH1 gen ekspresyon miktarları enzimatik
olarak belirlenmiştir. Tec1 proteininin yokluğunda NTH1 geninin aktive olduğu ve trehaloz birikiminin oldukça azaldığı belirlenmiştir. Elde edilen
sonuçlar Tec1 transkripsiyon faktörünün özellikle normal
üreme koşullarında NTH1
transkripsiyonunun negatif düzenlenmesi için gerekli olduğunu göstermiştir.

Anahtar Kelimeler

NTH1, Tec1p, trehaloz, Saccharomyces cerevisiae

Kaynakça

• Bao, M.Z., Schwartz, M.A., Cantin, G.T., Yates, J.R., Mdhani, H.D. 2004. “Pheromone-Dependent Destruction of the Tec1 Transcription Factor is Required for MAP Kinase Signaling Specificity in Yeast”, Cell, 119(7), 991-1000.
• Barbet, N.C., Schneider, U., Helliwell, S.B., Stansfield, I., Tuite, M.F., Hall, M.N. 1996. “TOR Controls Translation Initiation and Early G1 Progression in Yeast”, Molecular Biology of Cell, 7, 25–42.
• Beck, T., Hall, M.N. 1999. “The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors”, Nature, 402, 689–692. 16. Bell, W., Sun, W., Hohmann, S., Wera, S., Reinders, A., De Virgilio, C., Wiemken, A., Thevelein, J.M. 1998. “Composition and Functional Analysis of the Saccharomyces cerevisiae Trehalose Synthase Complex”, Journal of Biological Chemistry, 273(50), 33311-33319.
• Brückner, S., Kern, S., Birke, R., Saugar, I., Ulrich, H.D., Mösch, H.U., 2011. “The TEA Transcription Factor Tec1 Links TOR and MAPK Pathways to Coordinate Yeast Development”, Genetics, 189(2), 479-494.
• Cutler, N.S., Pan, X. Heitman, J., Cardenas, M. E. 2001. “The TOR Signal Transduction Cascade Controls Cellular Differentiation in Response to Nutrients”, Molecular Biology of Cell, 12, 4103–4113.
• Elbein, A.D., Pan, Y.T., Pastuszak, I., Carroll, D. 2003. “New Insights on Trehalose: a Multifunctional Molecule”, Glycobiology, 13(4),17R-27.
• Goldstein, A., Lampen, J.O., 1975. “Beta-D-Fructofuranoside Fructohydrolase from Yeast”, Methods in Enzymology, 42, 504- 511.
• Guarente, L., Ptashne, M., 1981. “Fusion of Escherichia coli lacZ to the Cytochrome c Gene of Saccharomyces cerevisiae”, Proceedings of the National Academy of Sciences, 78, 2199–2203.
• Heitman, J., Movva, N.R., Hall, M.N. 1991 “Targets for Cell Cycle Arrest by the Immunosuppressant Rapamycin in Yeast”, Science, 253, 905–909.
• Kyryakov, P., Beach, A., Richard, V.R., Burstein, M.T., Leonov, A., Levy, S., Titorenko, V.I. 2012. “Caloric Restriction Extends Yeast Chronological Lifespan by Altering a Pattern of Age-related Changes in Trehalose Concentration”, Frontiers in Physiology, 3, 256.
• Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. “Protein Measurement with the Folin Phenol Reagent”, Journal of Biological Chemistry, 193, 265-275.
• Madhani, H.D., Fink, G.R. 1997. “Combinatorial Control Required for the Specificity of Yeast MAPK Signaling”, Science, 275(5304), 1314-1317.
• Magasanik, B., Kaiser, C.A. 2002. “Nitrogen Regulation in Saccharomyces cerevisiae”, Gene, 290(1-2), 1-18.
• Parrou, J.L., François, J. 1997. “A Simplified Procedure for an Rapid and Reliable Assay of Both Glycogen and Trehalose in Whole Yeast Cells”, Analytical Biochemistry, 248, 186-188.
• Powers, R.W.3rd., Kaeberlein, M., Caldwell, S.D, Kennedy, B.K., Fields, S. 2006 “Extension of Chronological Life Span in Yeast by Decreased TOR Pathway Signaling”, Genes and Development, 20,174–184.
• Rose ,M.D., Winston, F., Heiter, P. 1990. “Methods in Yeast Genetics”. A Laboratory Course Manual, Cold Spring Harbor Laboratory Press Books.
• Schepers, W., Zebroeck, G.V., Pinkse, M., Verhaert, P., Thevelein, J.M. 2012. “In Vivo Phosphorylation of Ser21 and Ser83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase”, The Journal of Biological Chemistry, 287 (53), 44130-44142.
• Ter Schure, E.G., Van Riel, N.A., Verrips, C.T. 2000. “The Role of Ammonia Metabolism in Nitrogen Catabolite Repression in Saccharomyces cerevisiae”, FEMS Microbiology Reviews, 24: 67-83.
• Türkel, S, Turgut, T, López, M.C., Uemura, H., Baker, H.V. 2003. “Mutations in GCR1 affect SUC2 gene expression in Saccharomyces cerevisiae”, Molecular Genetics and Genomics, 268(6),825-831.