Expression of a mutated form of RABD2a, containing a 
single amino acid substitution in the conserved GTP binding motif, showed to be a dominant inhibitor, and revealed its role in targeting and fusion of ER-derived COPII vesicles at the Golgi surface. Recently, evidence for a Chlorhexidine hydrochloride chloroplast protein transport pathway involving the ER and Golgi apparatus in Arabidopsis has been presented. The carbonic anhydrase 1 protein was found to localize in the chloroplast stroma, despite its predicted ER signal peptide. Application of brefeldin A, a widely used fungal metabolite that interferes with Golgi-mediated vesicle traffic, obstructed transport of CAH1 to the chloroplast, causing it to arrest within the endomembrane system. The stromal protein was also shown to be N-glycosylated, confirming its transport via the endomembrane system to the chloroplast. Since then, other chloroplast proteins, including the rice a-amylase isoform I-1 and nucleotide pyrophosphatase/phosphodiesterase 1, were shown to follow the same or similar targeting pathways, indicating that several proteins might be transported to chloroplasts involving this pathway. Although no direct experimental evidence for the mechanism whereby the above mentioned plastid proteins are transported from the Golgi apparatus to the 4-(Benzyloxy)phenol plastids has been presented, trafficking from the ER to the Golgi, at least in monocot species, seems to depend on canonical elements such as ARF1 and SAR1. The incorporation of Golgi-resident proteins into plastids in both rice and onion cells appeared to be stimulated by expression of Amyl-1. These data suggest that communication between these compartments might be tightly regulated in vivo and that fine tuned expression of elements involved in vesicular trafficking and plastid N-glycoproteins must occur. While the Arabidopsis CAH1 protein harbours complex type Nglycans typical for proteins trafficking through the Golgi, the rice NPP1 and Amyl-1 seem to be modified with highmannose type N-glycans characteristic for the ER. Whether these differences reflect species-specific transport mechanisms remains to be clarified. Therefore, a molecular and genetic dissection of the elements involved in trafficking of these plastid glycoproteins is of great importance for our understanding of intracellular plant cell communication. To study the effect of dominant inhibitory GTPases on CAH1 trafficking, while avoiding secondary effects and lethality of the plant cells, we aimed to develop an experimental system for transient co-expression of such mutant proteins with CAH1. Transient expression techniques, such as protoplast transfection, usually results in a heterogeneous population of transfected/nontransfected cells. While transfection efficiency can vary from relatively low to significant, non-transfected cells will always be present and reduce or mask the effect on the total population as such. To circumvent this problem, fluorescent marker proteins are often fused to the protein of interest in order to enable visualization and analysis of the transfected cells. Unfortunately, previous studies on GTPases indicate that these proteins are sensitive to modifications, resulting in unstable forms with no activity when tagged at the N-terminus, and stable but with decreased activity when tagged at the C-terminus. One solution could be the use of the 2A peptide technology. The 2A peptide is a 16�C20 amino acid long peptide used by some RNA viruses for synthesis of multiple gene products from single transcripts.