The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis.

The ubiquitin receptors RPN10 and RPN13 harbor multiple activities including ubiquitin binding; however, solid evidence connecting a particular activity to specific in vivo functions is scarce. Through complementation, the ubiquitin-binding site-truncated Arabidopsis RPN10 (N215) rescued the growth defects of , supporting the idea that the ubiquitin-binding ability of RPN10 is dispensable and N215, which harbors a vWA domain, is fully functional. Instead, a structural role played by RPN10 in the 26S proteasomes is likely vital in vivo. A site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could partially rescue the growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with 26S proteasomes showed that the loss of RPN10 reduced the abundance of double-capped proteasomes, induced the integration of specific subunit paralogues, and increased the association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. Extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and UCH2-binding sites in the RPN13 C-terminus (246-254). Interestingly, an analysis of homozygous segregation in a background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13-RPN2 association in vivo.