Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli Identification of two cleavage sites

Abstract

The chloroplast transit pcptidc (cTP) of pea carbonic unhydrase was shown to be processed at two different sites, giving protein subunits of two sites. The cleavage sites were identiiied and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic rcsiducs, of the cTP. Properties of pea carbonic anhydrase produced in fkhcrichirr colishow that folding, oligomcrization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site T is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.

Kinetic studies of pea carbonic anhydrase

“…The enzyme appears on an SDS gel as a doublet corresponding to masses of 25 kDa and 27 kDa. In a previous study (Johansson and Forsman, 1992), we showed this doublet to represent subunits of two sizes present within pea chloroplasts. Calculation of the corresponding molecular masses from the published cDNA sequences (Roeske and Ogren, 1990;Majeau and Coleman, 1991) and N-terminal amino acid sequences from isolated subunits gives masses of 24.2 kDa and 28.2 kDa.…”

“…The inhomogeniety of the oligomers should also be considered in the interpretation of the CD spectra. The extra Nterminal part of the large subunit has the potential of forming an a-helix (Johansson and Forsman, 1992) and should contribute to the CD spectrum in the far-ultraviolet region. However, only a minor fraction of the subunits contain this extra N-terminal part.…”

“…It is nuclear-encoded and thus synthesised in the cytoplasm with a N-terminal chloroplast transit peptide, which is removed by a strornal peptidase after transport across the envelope membranes. In a recent study we have demonstrated two processing sites in the chloroplast transit peptide of pea CA, giving subunits differing by 4 kDa in size (Johansson and Forsman, 1992). The physiological role for chloroplast CA is not clearly understood, but it has been suggested to be important in generating CO, needed for carboxylation by ribulose-l,5-bisphosphate carboxylase (Reed and Graham, 1981).…”

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

“…Biochemical studies of pea (Pisum sativum) CA had previously identified cleavage of the chloroplast targeting sequence at two different sites, resulting in two different protein lengths (Johansson and Forsman, 1992). The respective predicted cleavage sites are located before and after a highly charged region of the protein corresponding to amino acid 107 in bCA1 (Johansson and Forsman, 1992). We analyzed the protein sequences of bCA1 expressed in tobacco leaves by western blot and identified a prominent protein band with an apparent M r of 25 kD ( Fig.…”

“…To further investigate this difference, we expressed different portions of bCA1 fused to YFPs in tobacco leaves. Biochemical studies of pea (Pisum sativum) CA had previously identified cleavage of the chloroplast targeting sequence at two different sites, resulting in two different protein lengths (Johansson and Forsman, 1992). The respective predicted cleavage sites are located before and after a highly charged region of the protein corresponding to amino acid 107 in bCA1 (Johansson and Forsman, 1992).…”