Two Arabidopsis Metacaspases AtMCP1b and AtMCP2b Are Arginine/Lysine-specific Cysteine Proteases and Activate Apoptosis-like Cell Death in Yeast

Naohide Watanabe,Eric Lam

doi:10.1074/jbc.m413527200

Abstract

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 (yca1Delta). Inducible expression of AtMCP1b and AtMCP2b significantly promoted yeast apoptosis-like cell death of both the wild-type and yca1Delta 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.

Highlights

Apoptosis or programmed cell death (PCD)1 is a conserved and genetically controlled process in multicellular organisms during development, homeostasis, and in response to pathogens and stress signals [1, 2]
To examine whether plant metacaspase genes are functionally related to yeast caspase-1 (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
When all metacaspases were individually expressed in the yca1⌬ mutant, a dominant band of about 47 kDa (AtMCP1b), kDa (AtMCP2b), and kDa (Yca1p), which likely correspond to the predicted molecular mass of each metacaspase tagged with a V5 epitope and hexahistidine (44.9, 46.7, and 52.4 kDa, respectively) were detected at 6 h after the galactose shift (Fig. 1A)

Summary

Functional Characterization of Arabidopsis Metacaspases

Initial execution phase for this developmental PCD [13]. direct biochemical evidence for the activity of the mcII-Pa encoded protein is lacking. The single metacaspase gene in S. cerevisiae, termed yeast caspase-1 (YCA1), was shown to mediate oxidative stress-induced and age-related apoptosis in yeast [19]. The wild-type Yca1p but not one with a point mutation at the catalytic center Cys297 residue was shown to be proteolytically activated upon oxidative stress, which correlated with the appearance of new caspase-like activities that have similar substrate specificity with initiator caspases (caspase-6 and caspase-8). Using the wild-type and yca1⌬ yeast strains, we performed a detailed analysis of the activities of AtMCP1b, AtMCP2b, and Yca1p during oxidative stress-stimulated and age-related apoptosis in yeast. We performed an initial biochemical characterization of these metacaspases produced in yeast and bacteria, to compare their enzymatic properties, including substrate preference

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION