Post-translational 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 reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated. SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted. The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.
Roberts
C S
CS
Department of Botany, Iowa State University, Ames 50011, USA.
Spalding
M H
MH
D000818:Animals; D002256:Carbonic Anhydrases / Q000096:biosynthesis* / Q000235:genetics; D002451:Cell Compartmentation; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D015151:Immunoblotting; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014026:Nicotiana / Q000235:genetics
D011994:Recombinant Proteins; D002256:Carbonic Anhydrases