Analysis of ER-Export of vacuolar transporter using the example of the V-ATPase in Arabidopsis thaliana
Final Report Abstract
The project dealt with the ER-export of the plant V-ATPase and the role of the Cornichon protein family in Arabidopsis thaliana. It was shown, that the V-ATPase localized transiently to ER export sites (ERES) that were labeled by the small GTPase Sar1. Further, the ER-export of membrane integral subunits of the V-ATPase was blocked and the subunits were arrested at the ERES, if the guanine nucleotide exchange factor Sec12 was overexpressed. These findings have proven the COP II-dependency of V-ATPases’ ER-export. In contrast to other subunits, the VHA-e1 subunit was constitutively found at ERES-like structures that were labeled by Sar1 and the Cornichon proteins, a family of putative cargo receptors. Neither VHA-E1 nor VHA-H had this effect on Sar1 although both showed a pattern typical for the ER. It was concluded that at least subunits of the membrane-integral sector, most probable VHA-e1, recruit Sar1 for ERES-formation. FRET-experiments as well as bimolecular fluorescence complementation revealed an interaction between VHA-e1 and members of the Cornichon-family, although the distribution of FRET-efficiencies was distinct for the isoforms 1, 3 and 4. Thus, VHA-e1 might function in mediating between the V-ATPase and the cargo receptors for efficient ER-export of the complex. Next, the mating-based split-Ubiquitin system was used to analyze the cargo-specificity of the Cornichon-family. Many interacting proteins were specific for only one member of the Cornichon-family so that the screen provides first evidence for a cargo-specificity of the family members. Combining the available data on protein-protein interactions in Arabidopsis thaliana in a Cytoscape network provided evidence for a crosstalk of distinct cargo-receptor families like p24s, CDC50s and the Cornichons.
