Functional characterization of coat protein i (copi) subunit isoforms in arabidopsis thaliana

Resumen

The early secretory pathway involves bidirectional transport between the endoplasmic reticulum (ER) and the Golgi apparatus and is mediated by coat protein complex I (COPI)- and coat protein complex II (COPII)-coated vesicles. COPII vesicles are involved in ER to Golgi transport while COPI vesicles mediate intra-Golgi transport and retrograde transport from the Golgi apparatus to the ER. The key component of the COPI coat is the coatomer complex, which is composed of seven subunits (α/β/β’/γ/δ/ε/ζ) and is recruited en bloc from the cytosol onto Golgi membranes. It has been described that the different COPI subunits participate in the biogenesis of the vesicles and in the selection of cargo that should be included in these vesicles. These two subunit functions are essential for cell integrity and viability. In contrast to mammals and yeast, two isoforms of β-COP subunit (β1- and β2-COP) and three isoforms of β´-COP subunit (β´1-, β´2- and β´3-COP) have been identified in Arabidopsis thaliana. This thesis aims to study the role of Arabidopsis β- and β´-COP subunit isoforms in plant biology. For this purpose, we have identified and characterized single and double loss-of-function mutants of all the genes that encode these isoforms. The results we have obtained suggest that Arabidopsis β1-COP and β2-COP genes may have redundant functions and that both β-COP genes are required for plant growth and tolerance to salt stress, specifically to chloride ions. In addition, β-COP function seems to be required for maintaining the structure of the Golgi apparatus. On the other hand, our data suggest that the three Arabidopsis β’-COP genes are at least partially redundant as none of the single β’-COP mutants displayed severe developmental defects under standard growth conditions. Nevertheless, β’-COP double mutant analysis indicates that β’3-COP cannot compensate for the simultaneous loss of β’1-COP and β’2-COP as no β’1β’2-cop double mutants could be obtained. Similarly, β’2-COP cannot compensate for the simultaneous loss of β’1-COP and β’3-COP since the β’1β’3-cop double mutant failed to develop beyond the seedling stage. However, β’2β’3-cop double mutants had no major phenotypic alterations, indicating that β’1-COP does seem to compensate for the simultaneous lack of β’2-COP and β’3-COP. These results appear to correlate with the seed development expression patterns of β’1-COP and suggest a role of β’1-COP during seed development that may affect seedling growth. Besides, it is also shown that the protein levels of α-COP are reduced in β´-COP double mutants. Finally, we have found that the trafficking of a canonical cargo of COPI vesicles, the p24 family protein p24δ5, is affected in β´-COP double mutants. This may be due to an impaired COPI-dependent Golgi-to-ER transport of p24δ5 that is mediated by its dilysine motif.