Chloroplasts import many preproteins that can be classified based on their physicochemical properties. The cleavable N-terminal transit peptide (TP) of chloroplast preproteins contains all the information required for import into chloroplasts through Toc/Tic translocons. The question of whether and how the physicochemical properties of preproteins affect TP-mediated import into chloroplasts has not been elucidated. Here, we present evidence that Pro residues in TP mediate efficient translocation through the chloroplast envelope membranes for proteins containing transmembrane domains (TMDs) or proteins prone to aggregation. By contrast, the translocation of soluble proteins through the chloroplast envelope membranes is less dependent on TP prolines. Proless TPs failed to mediate protein translocation into chloroplasts; instead, these mutant TPs led to protein mistargeting to the chloroplast envelope membranes or nonspecific protein aggregation during import into chloroplasts. The mistargeting of TMD-containing proteins caused by Pro-less TPs in wild-type protoplasts was mimicked by wild-type TPs in protoplasts, in which preprotein translocation is compromised. We propose that the physicochemical properties of chloroplast proteins affect protein translocation through the chloroplast envelope, and prolines in TP have a crucial role in the efficient translocation of TMD-containing proteins.

Carbonic anhydrase (CA; EC 4.2.2.1) is a Zn-binding metalloenzyme that catalyzes the reversible hydration of CO 2. Recently, CAs have gained a great deal of attention as biocatalysts for capturing CO 2 from industrial flue gases owing to their extremely fast reaction rates and simple reaction mechanism. However, their general application for this purpose requires improvements to stability at high temperature and under in vitro conditions, and reductions in production and scale-up costs. In the present study, we developed a strategy for producing GcCAα3, a CA isoform from the red alga Gracilariopsis chorda, in Nicotiana benthamiana. To achieve high-level expression and facile purification of GcCAα3, we designed various constructs by incorporating various domains such as translation-enhancing M domain, SUMO domain and cellulosebinding domain CBM3. Of these constructs, MC-GcCAα3 that had the M and CBM3 domains was expressed at high levels in N. benthamiana via agroinfiltration with a yield of 1.0 g/kg fresh weight. The recombinant protein was targeted to the endoplasmic reticulum (ER) for high-level accumulation in plants. Specific and tight CBM3-mediated binding of recombinant GcCAα3 proteins to microcrystalline cellulose beads served as a means for both protein purification from total plant extracts and protein immobilization to a solid surface for increased stability, facilitating multiple rounds of use in CO 2 hydration reactions.