Abstract
Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box (PUB) armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.
Highlights
Post-translational modifications of proteins regulate their function and abundance
For SAUL1 it has been demonstrated that this specific localization depends on the additional ARM repeat domain in the C-terminus that is unique to SAUL1, AtPUB42, and AtPUB43
To test whether the C-terminal ARM repeat domain is essential for plasma membraneassociation of AtPUB43, we analyzed the localization of fusion proteins between GFP and truncated AtPUB43 proteins by confocal laser scanning microscopy on transformed Arabidopsis protoplasts
Summary
Post-translational modifications of proteins regulate their function and abundance. Ubiquitination of target proteins, in which the C-terminus of ubiquitin is covalently bound to other proteins and attached to itself in growing ubiquitin chains, modifies protein function in case of mono-ubiquitination and triggers degradation of poly-ubiquitinated proteins via the 26S proteasome. E3 ubiquitin ligases are crucial for attachment of ubiquitin to target proteins that are recognized. This specific ubiquitination of proteins via E3 ubiquitin ligases requires the preceding activation of ubiquitin by E1 ubiquitin-activating enzymes and the subsequent transfer to E2 ubiquitin-conjugating enzymes. Plant U-box (PUB) proteins represent the most recently identified type of E3 ubiquitin ligases. In Arabidopsis, the group of PUB proteins comprises 64 members (Azevedo et al, 2001; Wiborg et al, 2008). These PUB proteins contain the highly conserved U-box that is required for E2 binding (Pringa et al, 2001). Most of the members contain armadillo (ARM) repeats, which likely constitute interfaces for protein-protein interactions, and are named
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