Cloning and characterization of the calreticulin gene from Ricinus communis L.

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase. Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3'-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5'-untranslated genomic sequence fused to the beta-glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.

Molecular characterisation of plant endoplasmic reticulum. Identification of protein disulfide-isomerase as the major reticuloplasmin.

Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase. Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein.