Heterologous expression of the tomato protein Asr1 in yeast cells brings evidence on its biological function

NICOLÁS URTASUN; RAFAEL ARGÜELLO; SUSANA CORREA GARCÍA; NORBERTO D. IUSEM; MARIANA BERMUDEZ MORETTI

Toronto

Congreso; Yeast genetics & molecular biology meeting; 2008

The Genetics Society of america

Asr1, a gene induced by stress in plants, belongs to a family composed by at least four members. The biochemical function of the encoded protein remains unknown although two alternative and not mutually exclusive possible roles have been postulated: 1) protective against water loss, based on its high degree of hydrophylicity. 2) transcription factor, from evidence on nuclear localization and DNA-binding activity. To test the possible mechanism of ASR1 in protecting cells from water loss, the protein tomato was expressed in the heterologous expression system Saccharomyces cerevisiae under control of a galactose-inducible promoter. We have earlier demonstrated that ASR1 complements the hyperosmotic sensitivity of mutants deficient in the HOG (High Osmolarity Glycerol) pathway. To validate our experimental design, we analysed by flux cytometry (FACS) the expression of the GFP reporter gene driven by the osmotic stress-responsive Hsp12 promoter when Asr1 was overexpressed. We made sure that the accumulation of Asr1 within the cells did not trigger any stress response. We showed that Asr1 led to the accumulation of glycerol in HOG-deficient strains. These results suggest that Asr1 acts as a positive transcription factor specific for genes involved in glycerol accumulation. However, our preliminary results using confocal microscopy indicated that Asr1 is mainly located in the cytoplasm. The expression of several truncated ASR1 proteins gave us important information about the mechanism by which ASR1 protects yeast cells from osmotic stress.