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Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein
M Tokunaga1, A Kawamura, F Hishinuma
1Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
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 with that of 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.
Insights
The 28K subunit of yeast killer toxin kills host cells internally when expressed. Killer immunity prevents this cell death, confirming the 28K subunit
Area of Science:
- Molecular Biology
- Yeast Genetics
- Protein Expression
Background:
- Yeast killer toxins are secreted proteins encoded by plasmids pGKL1 and pGKL2 in Saccharomyces cerevisiae.
- The killer toxin complex comprises 97K, 31K, and 28K subunits.
- The precise role and localization of individual subunits in yeast cell killing remain to be fully elucidated.
Purpose of the Study:
- To investigate the role of the 28K subunit in yeast killer toxin activity.
- To determine the localization and mechanism of action of the expressed 28K subunit.
- To assess the contribution of the 28K subunit to host cell death.
Main Methods:
- Cloning the 28K subunit gene into a circular plasmid with a modified promoter for expression in Saccharomyces cerevisiae.
- Expressing the 28K subunit in killer-sensitive yeast strains.
- Co-expressing killer immunity factors to assess their protective effect.
Main Results:
- The 28K subunit was successfully expressed in S. cerevisiae.
- Expression of the 28K subunit led to the death of host cells.
- Host cell death was prevented by the expression of killer immunity, implicating the 28K subunit in the killing activity.
- The 28K subunit precursor, despite having a signal sequence, was retained intracellularly, not secreted.
- This intracellular retention suggests the 28K subunit kills cells from within.
Conclusions:
- The 28K subunit is the primary determinant of the killer toxin's cytotoxic activity.
- The 28K subunit mediates cell death intracellularly, rather than through extracellular secretion.
- The absence of the 97K and 31K subunits does not impair the 28K subunit's intracellular killing ability.