Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B

Isolation and Characterization of Protein Bodies in Lupinus angustifolius

Using Nycodenz, a novel density gradient medium, we isolated intact protein bodies from developing seeds of Lupinus angustifolius L. (cultivar Unicrop) and achieved excellent separation from the endoplasmic reticulum, mitochondria, and other organelles. The distribution of the storage protein conglutin-β was taken as evidence that up to 96% of the protein bodies remained intact on the gradients and banded at 1.25 grams per milliliter. The protein bodies also contained the three other abundant proteins present in L. angustifolius seeds: conglutins-α, -γ, and -δ. Pulse labeling experiments were carried out to determine the site of proteolytic processing of conglutin-α, a legumin-like 11Svedberg unit storage protein. Cotyledons aged either 33 or 40 days after flowering were pulsed with [(3)H]leucine. Protein bodies obtained from the cotyledons aged 33 days after flowering contained only the labeled precursors of conglutin-α with molecular weights 85,000, 72,000, and 64,000, even after a 4 hour chase of the radioactivity. Protein bodies obtained from the cotyledons aged 40 days after flowering contained the same radioactive precursors if the tissue had been pulsed for 2 hours, and the processing products of these precursors when the tissue had been chased for 4 hours. These studies confirm that the subcellular location of proteolytic cleavage of this legumin-like protein is the protein body, that this activity is detected only in protein bodies from lupin seeds aged between 33 and 40 days of seed development after flowering and that protein bodies from seeds younger than this contain only unprocessed conglutin-α