Abstract
Development of the aleurone layer of maize grains requires the activity of the Defective kernel 1 (Dek1) gene, encoding a predicted 240-kDa membrane-anchored protein with a C terminus similar to animal calpain domain II&III. Three-dimensional modeling shows that DEK1 domain II contains a conserved calpain catalytic triad and that domain II&III has a predicted structure similar to m-calpain. Recombinant DEK1 domain II&III exhibits activity in the caseinolytic assay in the absence of calcium, although the activity is enhanced by calcium. This is in sharp contrast to animal calpains, which require Ca2+ to be active. Bacterially expressed DEK1 domain II does not display caseinolytic activity, suggesting an important role for DEK1 domain III. Mutation of the catalytic Cys residue to Ser leads to a loss of caseinolytic activity of DEK1 domain II&III. Two features of DEK1 calpain may contribute to maintaining the active site triad in an "active" configuration in the absence of Ca2+, both of which are predicted to keep m-calpain domains IIa and IIb apart. First, DEK1 lacks key charged residues in the basic loop of domain II, and secondly, the absence of an acidic loop in domain III, both of which are predicted to be neutralized upon Ca2+ binding. The Dek1 transcript is present in all cell types in developing maize endosperm, suggesting that the activity of the DEK1 calpain is regulated at the post-transcription level. The role of DEK1 in aleurone signaling is discussed.
Highlights
Calpains are a family of Ca2ϩ-dependent cytosolic cysteine proteinases [12, 13] that consist of ubiquitous and tissue-specific isoforms in animals
Recombinant DEK1 calpain is active in the caseinolytic assay previously used for animal calpains, and this activity is abolished by changing the cysteine of the active site triad to serine (Figs. 4 – 6)
Regulation in vivo of conventional animal calpains such as m-calpain is complex, and operates at several levels, including signal transduction, Ca2ϩ activation, subcellular relocation from cytosol to, or near, the plasma membrane, interaction with phospholipids, autocatalytic cleavage, and interaction with a regulatory small subunit
Summary
Molecular Modeling of DEK1 Calpain Domains—The crystal structure of rat (Rattus norvegicus) m-calpain catalytic subunit (PDB 1DF0_A, 2.6 Å resolution) was used as a template to model the calpain domain of DEK1. After insert sequences were verified by sequencing, the constructs were transformed into BL21 (DE3) E. coli (Invitrogen) for protein expression. The Expression of DEK1 Domain II—E. coli BL21 cell cultures were grown in LB medium (Difco Laboratories) with 100 g/ml carbenicillin. The transformed cells harboring Dek domain II were grown at 37 °C to an absorbance of ϳ1.0 at 600 nm. The cells were harvested for the GST-DEK1 domain II purification as mentioned above. Site-directed Mutagenesis of DEK1—Site-directed mutagenesis of DEK1 domain protein was generated by a PCR-based overlapping method described by Ho et al [33]. A typical reaction contained 2 g of DEK1 domain protein, 3 g of purified -casein (Sigma-Aldrich C6905, more than 90% purity) in 20 l of reaction buffer (50 mM imidazole-HCl, pH 7.5, 10 mM -mercaptoethanol). Microscopy evaluation was carried out in dark field using a Nikon Eclipse E800 microscope
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