Abstract
BackgroundA tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16–20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited.Methodology/Principal FindingsThe aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system.Conclusions/SignificanceWe demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.
Highlights
The plant endomembrane system is a highly dynamic structure and comprises several biochemically distinct membrane-bound organelles that are linked by membrane traffic
Transient expression of such mutants has shown that SAR1 controls the assembly of the protein COPII coat that direct vesicle budding from endoplasmic reticulum (ER), whereas the ARF1 GTPase performs a similar function for Golgiderived COPI vesicle budding as well as post-Golgi traffic [5,6,7]
To continue our study of the mechanisms involved in transport of carbonic anhydrase 1 (CAH1) between the ER and the Golgi, we explored the requirement for specific GTPases involved in vesicle trafficking
Summary
The plant endomembrane system is a highly dynamic structure and comprises several biochemically distinct membrane-bound organelles that are linked by membrane traffic. Raslike small GTP-binding proteins, such as SAR1, ARF1, and RAB GTPases, play an important role in the general mechanism of vesicular trafficking pathways in all eukaryotic cells [1,2] They share a common structure and act as molecular switches by cycling between active GTP-bound and inactive GDP-bound states [1]. The use of negative mutants that are locked at the GTP- or GDPbound state, acting dominantly over the wild type proteins, has advanced the understanding of their function in plant intracellular vesicular trafficking [2,3,4] Transient expression of such mutants has shown that SAR1 controls the assembly of the protein COPII coat that direct vesicle budding from endoplasmic reticulum (ER), whereas the ARF1 GTPase performs a similar function for Golgiderived COPI vesicle budding as well as post-Golgi traffic [5,6,7]. The use of the 2A technology in plant systems has been limited
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