Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae

Abstract

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I− and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription. The WT cells, under I− significantly reduced the expression of GCR1 and PIS1, but increased the expression of KCS1 and de-repressed INO1. The Kcs1p expression was reduced in gcr1∆ cells; this reduced INO1 expression resulting in abnormal vacuolar structure and reduced autophagy in Saccharomyces cerevisiae.

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Acknowledgements

This work was supported by the Science and Engineering Research Board (SERB) Grant No: EMR/2016/001727, New Delhi, under EMR scheme. C. Ravi was supported by a fellowship from SERB, New Delhi. We are grateful to Prof. Ram Rajasekharan (Central Food Technological Research Institute, Mysore, India) for providing yeast strains, over expression plasmids, reagents and instrument facility. We thank Prof. Rashna Bhandari (Centre for DNA Fingerprinting and Diagnostics, India), and John M. Lopes (College of Natural Sciences, University of Massachusetts, Amherst, MA) for providing pYES2-KCS1 and YEp357R-INO1-LacZ plasmids. We thank Prof. Tracy L. Johnson (Molecular Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA), Prof. Ji-Sook Hahn (School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea), and Prof. Ravi Manjithaya (Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research Jakkur, Bangalore 560 064, India) for providing plasmids pRS315-GCR1-TAP tag, pRS415GPD, pRS415GPD-GCR1 and pRS316–ATG8–GFP, respectively. We thank Prof, Yoshinori Oshsumi (Tokyo Institute of Technology, Yokohama, Japan) for providing Ape1 antiserum. We are thankful for the infrastructure facilities of DST-FIST, Department of Biochemistry, Life Sciences and DST-PURSE facilities, of Bharathidasan University.

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V.N. and C.R designed the experiments. C.R and R.G performed the experiments. C.R and V.N. discussed the data and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

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Correspondence to Vasanthi Nachiappan.

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Ravi, C., Gowsalya, R. & Nachiappan, V. Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae. Curr Genet 65, 995–1014 (2019). https://doi.org/10.1007/s00294-019-00954-2

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Keywords

  • Phosphatidylinositol
  • Inositol
  • PIS1
  • KCS1
  • Autophagy