biostudies-literatureUnknown25(21)Lin SYThe 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 <i>rpn10-2</i>, 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 <i>rpn10-2</i> growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with <i>rpn10-2</i> 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 <i>rpn10-2</i> segregation in a <i>rpn13-1</i> background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13-RPN2 association in vivo.International journal of molecular sciences11650https://www.ebi.ac.uk/biostudies/studies/S-EPMC11546751biostudies-literatureThe Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis.PMC11546751Fu HLin YLUsharani RRadjacommare RLin SYfalseThe 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 <i>rpn10-2</i>, 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 <i>rpn10-2</i> growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with <i>rpn10-2</i> 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 <i>rpn10-2</i> segregation in a <i>rpn13-1</i> background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13-RPN2 association in vivo.2024-01-01T00:00:00Z2024 Oct2026-07-12T03:13:15.308Z2025-04-04T18:55:41.002ZS-EPMC115467513951920710.3390/ijms252111650