POSTTRANSLATIONAL PROCESSING OF THE HIGHLY PROCESSED, SECRETED PERIPLASMIC CARBONIC-ANHYDRASE OF CHLAMYDOMONAS IS LARGELY CONSERVED IN TRANSGENIC TOBACCO

The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas rei nhardtii codes for a highly processed secreted glycoprotein. The prima ry translation product of the CAH1 gene is targeted to the ER, where i t is proteolytically processed to yield two different subunits, glycos ylated, assembled into an active heterotetramer, and secreted. After r eplacing the target leader sequence with that from tobacco anionic per oxidase, expression of this gene in transgenic tobacco plants was inve stigated. SDS-PAGE gels of the purified protein from tobacco, showed t hat it migrated as a series of discrete bands (two large and one small ) with slightly faster mobility than the comparable bands in the purif ied algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glyc osylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was s ecreted. The specific activity of the enzyme and its sensitivity to su lfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamy domonas peri-CA1 is largely conserved in a higher plant.