The Calpain Domain of the Maize DEK1 Protein Contains the Conserved Catalytic Triad and Functions as a Cysteine Proteinase

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 Ca 2؉ 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 Ca 2؉ , 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 Ca 2؉ 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 posttranscription level. The role of DEK1 in aleurone signaling is discussed.In cereal grains, the aleurone layer consists of densely cytoplasmic cells covering the surface of the endosperm, the grain storage tissue that is used for feed, food and industrial raw material (1). Aleurone cells contain a large numbers of protein and oil bodies and are cytologically and biochemically distinct from the storage cells of the underlying starchy endosperm. Upon imbibition of the grain, aleurone cells secrete enzymes that mobilize stored starch and protein reserves for seedling growth (2). The endosperm develops from the central cell after double fertilization through a cellularization process that results in a peripheral layer of aleurone cell initials (3). From genetic evidence, the tumor necrosis factor receptor-like kinase CRINKLY4 (CR4) is implicated in aleurone cell fate specification (4 -7). Based on the identity of the Cr4 gene product, as well as the peripheral position of the aleurone layer, we proposed a model in which the CR4 receptor is activated by a ligand in the periphery of the endosperm (8). Recently, we isolated the defective kernel 1 (dek1) 1 gene, which is also essential for aleurone cell development (9). Homozygous recessive dek1 endosperm initiates aleurone cell fate specification, but fails to maintain aleurone cell fate, resulting in grains that lack aleurone cells (9). Revertant sector analysis has demonstrated that Dek1 function is essential for the maintenance of aleurone cell fate throughout grain development (10, 11). Sequence analysis predicts that the Dek1 gene encodes a 240 kDa protein with 21 membrane spa...

Subcellular Localization and Functional Domain Studies of DEFECTIVE KERNEL1 in Maize andArabidopsisSuggest a Model for Aleurone Cell Fate Specification Involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1

“…This hypothesis was based on the highly conserved active site of DEK1 compared with typical animal calpains as well as on the fact that DEK1-CALP displays calpain Cys proteinase activity in vitro (Wang et al, 2003). Here, we have shown that maize DEK1-CALP failed to complement the loss-of-aleurone cell phenotype in homozygous maize dek1 mutant endosperm under the control of the 27-kD g-Zein promoter.…”

“…Although the three-dimensional structure of DEK1 is unknown, it has been speculated that the predicted extracellular portion of the molecule interacts with external ligands or membrane proteins representing positional information. Furthermore, we have proposed that external signals perceived by DEK1-MEM are transmitted via the calpain-like Cys protease (DEK1-CALP) predicted to be in the cytosol ( Figure 1A) (Wang et al, 2003). Third, DEK1 is ubiquitously expressed in the endosperm, fulfilling an important requirement for a protein involved in sensing and/or transmitting cell surface positional information (Lid et al, 2002).…”

The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo

“…Finally, partial inhibition of the proteolysis by EGTA is indicative of a calcium-stimulated cysteine protease activity such as the maize DEK1, which belongs to the calpain family (27). DEK1 is a membrane-bound protein that contains a calpain catalytic triad, and it displays high structure homology in domains II and III to the animal small opticlobes (SOL), and SOHL (small homolog from human) calpains (28). In contrast to the animal calpains, DEK1 exhibits strong proteolytic activity in the absence of calcium that increases further in the presence of Ca 2ϩ , similar to what we observed in this study (Fig.…”

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize

“…Homozygous cr4 endosperms lack patches of aleurone cells, and leaves of homozygous mutant plants are crinkly. Dek1 encodes a protein with 21 predicted transmembrane segments and a calpain-like protease in the C terminus (Lid et al, 2002;Wang et al, 2003). Homozygous dek1 endosperms almost entirely lack aleurone cells, and embryos are arrested at the globular stage of development (Lid et al, 2002).…”