Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast

Metacaspases in plants, fungi, and protozoa constitute new members of a conserved superfamily of caspase-related proteases. A yeast caspase-1 protein (Yca1p), which is the single metacaspase in Saccharomyces cerevisiae, was shown to mediate apoptosis triggered by oxidative stress or aging in yeast. To examine whether plant metacaspase genes are functionally related to YCA1, we carried out analyses of AtMCP1b and AtMCP2b, representing the two subtypes of the Arabidopsis metacaspase family, utilizing yeast strains with wild-type and the disrupted YCA1 gene (yca1 Delta). Inducible expression of AtMCP1b and AtMCP2b significantly promoted yeast apoptosis-like cell death of both the wild-type and yca1 Delta strains, relative to the vector controls, during oxidative stress and early aging process. Mutational analysis of the two AtMCPs revealed that their cell-death-inducing activities depend on their catalytic center cysteine residues as well as caspase-like processing. In addition, the phenotype induced by the expression of two AtMCPs was effectively prevented when the cells were pretreated with a broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone. These results suggest that the two subtypes of Arabidopsis metacaspases are functionally related to Yca1p with caspase-like characteristics. However, we found that bacterial and yeast extracts containing AtMCP1b, AtMCP2b, or Yca1p exhibit arginine/lysine-specific endopeptidase activities but cannot cleave caspase-specific substrates. Together, the results strongly implicate that expression of metacaspases could result in the activation of downstream protease(s) with caspase-like activities that are required to mediate cell death activation via oxidative stress in yeast. Metacaspases from higher plants may serve similar functions.