Expression of pGKL killer 28K subunit inSaccharomyces cerevisiae: identification of 28K subunit as a killer protein

Abstract

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15 1031-1046 (1987)). The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced withthatof S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.