Genetic analysis of growth inhibition of yeast cells caused by expression of Aspergillus oryzae RNase T1

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promot er with a single-copy vector were able to grow on galactose medium while th ose with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affe ct the growth. We also observed that expression of RNase T1 without a signa l sequence severely inhibited growth of the transformant even on the single -copy plasmid. Subcellular fractionation showed that overexpressed myc-tagg ed RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expr ession of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secre tory apparatus has toxic effects on the host cells.