Expression and purification of plant proteins for functional studies Expression of epitope-labeled carbonic anhydrase in Arabidopsis plants

Chloroplasts are cell organelles responsible for photosynthesis. Although chloroplast have their own genome it is not sufficient to encode all the proteins which are located there. Most of the proteins are imported from the cytosol through the so called toc/tic pathway. It has been recently showed that Arabidopsis CAH1 is transported to the chloroplast through the secretory route in a fully new pathway. It has also been demonstrated that the N-terminal signal peptide of CAH1 targets it to the ER where the protein gets glycosylated. Structure of the Arabidopsis CAH1 suggests that its Cterminus might be responsible for targeting the protein to the chloroplast. By expressing Nand C-terminal labeled CAH1 we show that the expression level of the N-terminal labeled form is high and the majority of the labeled protein is localized in the chloroplast. By contrast, the C-terminal labeled CAH1 expressed weakly if at all, and due to the low expression level immunolocalization of the protein is difficult. We also demonstrate that the strong expression level of the N-terminal labeled CAH1 makes it feasible to affinity purify the glycosylated protein for structural studies. Chloroplasts are cell organelles responsible for photosynthesis which are located in plant cells and eukaryotic algae. The structure of chloroplast is almost the same as cyanobacteria (blue algae). Chloroplast like cyanobacteria have a nonchromosomal structured DNA-genome and they have their own protein synthesis. The chloroplast ribosomes are from 70S type like by prokaryotes. Further the chloroplast ribosomal proteins show a structural similarity to those in cyanobacteria. All this support the endosymbiotic theory which stipulates that chloroplasts were cyanobacteria which have been ingested by a cell. The result of this event was the loss of a large amount of DNA which is supposed to have been transported to the nucleus. This means that over 90% of the proteins which are found in the chloroplast are synthesised in the cytosol and then transported to the chloroplast. Thus a process has been developed to transport cytosol synthesized proteins to the chloroplast. It is generally accepted that the cytosol synthesized pre-proteins are targeted to the chloroplast by their transit sequence which is located at the Nterminus of the pre-protein. This transit peptide interacts with a complex which is located in the chloroplast membrane. The so called toc/tic (translocon outer/inner membrane) complex allows the entrance of the protein into the chloroplast and so the import of the protein into the stroma (Jarvis and Soll, 2001). The Arabidopsis CAH1 belongs to the carbonic anhydrase family. This zinc enzyme family, which is divided in 3 groups ( , and ) catalyses the hydratation of CO2 to H2CO3. The structure of CAH1 shows that it carries a signal peptide at its N-terminal and a lysine cluster at its C-terminal (highly hydrophobe). This lysine cluster is a conserved region in -carbonic anhydrase from other organisms. The C-terminal of the Arabidopsis CAH1 has some similarities with the acyl carrier protein of the malaria parasite Plasmodium falciparum (Foth et al., 2003) which needs lysine for targeting to the apicoplast of the parasite. This suggests that the C-terminal might also play a role in the targeting of CAH1 to the chloroplast. CAH1 also carries five putative glycosylation sites which have not been defined yet. It has been previously showed by immunolocalization analysis and biochemical fractionation that the Arabidopsis carbonic anhydrase CAH1 (accession number AAC32523) is located in the chloroplast stroma. CAH1 is predicted to enter the secretory pathway by neural network-based tool TargetP (Emanuelsson et al., 2000 and Nielsen et al., 1997). Arabidopsis CAH1 appears to be transported differently from the common accepted protein transport pathway. It has been showed that Arabidopsis CAH1 is targeted to the chloroplast through the secretory pathway (Villajero et al., 2005). Indeed they have demonstrated that the Arabidopsis CAH1 is targeted by its signal peptide to the ER (endoplasmatic reticulum) where the signal peptide is cleaved off. Also the N-glycosylation of the protein starts there before it is transported to the Golgi. The Nglycosylation of CAH1 is completed in the Golgi then the protein is either directly transported to the chloroplast or indirectly to the chloroplast (Villajero et al., 2005). The N-glycosylation of proteins has a great impact on their physico-chemical and biological function. The N-glycosylation of protein in plants starts with the transfer in the ER (endoplasmatic reticulum) of the oligosaccharide precursor Glc3Man9GlcNAc2 from a dolichol lipid carrier to specific Asparagine (Asn) residues on the nascent polypeptide chain (Rayon, Lerouge and Faye, 1998). After the transfer, Glc3Man9GlcNAc2 undergoes trimming of the glucose (Glc) and some mannose (Man) residues, first in the ER and then in the Golgi, giving rise to high–mannose-type N-glycans from five to nine mannose residues (Maia and Leite, 2001). Plant N-glycans can be further modified in the Golgi into complex-type N-glycans during the transport of glycoprotein from the cis, through medial to trans cisternae (Lerouge and Faye, 1998). N-glycans can still be modified after their maturation in the ER and the Golgi either during their transport or in the compartment of its final location. Some glycoproteins in bacteria are glycosylated in the chloroplasts (Upreti et al., 2003) but this doesn’t happen with CAH1 since its glycosylation goes through the secretory system. N-glycosylation of proteins prevents them against proteolytic degradation and plays a role in the correct folding and biological activity of the protein. It has also been suggested they might contain targeting information or are directly involved in protein recognition or cell-cell adhesion pathway. The aim of this project was to express Nand C-terminal labeled CAH1 proteins in Arabidopsis cells, analyse their expression level and localisation and finally, to purify the expressed protein for structural studies. To achieve these goals we first fused a HA-SBP (Hemagglutinin-Streptavidin Binding Peptide) tandem-epitope tag to the Nand the C-terminal of the CAH1. This gave us Nand C-labeled CAH1 constructs to analyse the role of the Cand the Nterminal of CAH1. Then the constructs were transfected into Arabidopsis protoplasts and the protein expression were analysed by immunoblotting. This allowed us to conclude which epitope-labeled CAH1 is expressed or better expressed in the Arabidopsis protoplasts . We then made immunolocalization of both Cand N-terminal epitope-labeled CAH1 in Arabidopsis protoplasts to determine, by confocal microscopy, where in the cell the protein is located. We used protoplasts from the Arabidopsis Columbia and the Erecta ecotypes since the expression of protein is better in the first one but the second one is more suitable for confocal analysis. Since we also intended to analyse the expression of the proteins in vivo, we wanted to create constructs in a binary vector and then transform Arabidopsis plants. Finally the extracted proteins should be purified and analised by mass spectrometry.