Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1.

The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences.

The abscisic acid (ABA)-induced protein kinase PKABA1 is present in dormant seeds and is a component of the signal transduction pathway leading to ABA-suppressed gene expression in cereal grains. We have identified a member of the ABA response element-binding factor (ABF) family of basic leucine zipper transcription factors from wheat (Triticum aestivum) that is specifically bound by PKABA1. This protein (TaABF) has highest sequence similarity to the Arabidopsis ABA response protein ABI5. In two-hybrid assays TaABF bound only to PKABA1, but not to a mutant version of PKABA1 lacking the nucleotide binding domain, suggesting that binding of TaABF requires prior binding of ATP as would be expected for binding of a protein substrate by a protein kinase. TaABF mRNA accumulated together with PKABA1 mRNA during wheat grain maturation and dormancy acquisition and TaABF transcripts increased transiently during imbibition of dormant grains. In contrast to PKABA1 mRNA, TaABF mRNA is seed specific and did not accumulate in vegetative tissues in response to stress or ABA application. PKABA1 produced in transformed cell lines was able to phosphorylate synthetic peptides representing three specific regions of TaABF. These data suggest that TaABF may serve as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy expression, and ABA-suppressed gene expression.