Although use of 2A technology has previously been reported in plants, Samalova and coworkers have questioned its use in plant systems, demonstrating that further development and characterization of the 2A system is necessary. In this study, we wanted to improve the 2A technology for use in plant cells as a tool to study basic cellular processes, such as protein trafficking mechanisms. After successful optimization of 2A cleavage efficiency, mutant forms of small GTP-binding proteins could be expressed and used to interfere with trafficking of CAH1 at the endomembrane system. The results presented in this work highlight the 2A technology as a valuable tool for effective and stoichiometric co-expression of marker and effector molecules in plant systems. In addition, our study demonstrates that 2A mediated transient co-expression of fluorescent markers combined with fluorescent activated cell sorting can be used to obtain homogeneous mutant protoplast populations in very short time. Our data shows that similar to RABD2a, dominant mutant versions of SAR1 and ARF1 originating from 
the GSCFP-2A construct were functional and gave a phenotypic effect on the Golgi-localized marker protein. The effects were consistent to those observed in previous studies. Our findings clearly demonstrate the value of a stoichiometric co-expression system that can easily be applied to live-cell imaging and confocal microscopy studies. These results prompted us to further extend and test the versatility of the 2A system in combination with other experimental techniques, such as biochemical assays and fluorescence-activated cell sorting. To Mechlorethamine hydrochloride continue our study of the mechanisms involved in transport of CAH1 between the ER and the Golgi, we explored the requirement for specific GTPases involved in vesicle trafficking. Although BFA arrested CAH1-GFP in aggregate-like structures, it is known to have different effects in different species, and even in different tissues within a species. In addition, high BFA concentrations likely induce secondary effects, alterations that can be reduced using genetic tools. The potential involvement of three individual GTPases was tested in this initial study; RABD2a, SAR1, and ARF1, all of them important for vesicle formation or docking at the ER-Golgi interface. Specific single point mutations in these GTPases can generate arrested and non-functional enzymes. RABD2a is a small GTPase involved in targeting and fusion of ER-derived COPII vesicles at the Golgi surface. Dominant negative variants where an N121I substitution was introduced in the GTP binding motif ), were shown to inhibit trafficking of secreted and Golgi targeted proteins out from the ER. On the contrary, SAR1 and ARF1 are directly involved in the formation of COPII and COPI vesicles, respectively. Two mutant Chlorhexidine hydrochloride isoforms of ARF1 were tested: ARF1 and ARF1, both affecting ER-to-Golgi trafficking and relocating Golgi markers to the ER. The ARF1 mutant shows reduced GTPase activity, therefore acting as a constitutively activated mutant, interfering with sorting of membrane proteins into Golgiderived COPI vesicles. The ARF1 mutant instead has low affinity for GTP, acting as a dominant-negative mutant that blocks formation of COPI vesicles. We induced ARF1 expression under the control of a heat-shock promoter in fourweeks-old Arabidopsis plants. Unfortunately, ARF1 expression in leaf tissue was very low, which in combination with the time needed for obtaining good chloroplast preparations, prompted us to seek a different experimental system.