[Hongyong Fu] Non-ubiquitin binding functions for two major ubiquitin receptors RPN10 and RPN13 from Arabidopsis
POST:
The RPN10 D11 residue critical for the 26S proteasome structural stability and the RPN13 F67 residue critical for RPN2 binding are important in vivo.
Although it is well perceived that conserved ubiquitin receptors play a vital role in recognition and targeting ubiquitinated cellular regulators to proteasome for degradation, solid evidence to support the in vivo importance of ubiquitin-binding activities for these ubiquitin receptors in vivo is scarce. Dr. Hongyong Fu’s group reported previously the ubiquitin binding activities of the major ubiquitin receptor RPN10 is dispensable for in vivo functions observed with rpn10-2 null mutant. A new report just published in Int J Mol Sci from Dr. Fu’s group confirms this idea again by observing that a C-terminal truncated RPN10 harboring only a vWA domain and missing all ubiquitin binding motifs is fully functional in vivo. Instead, all evidence provided in this report support RPN10 plays an important structural role in the 26S proteasome in vivo. First, a site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could only partially rescue the rpn10-2 growth defects and is not integrated into 26S proteasomes. Second, native polyacrylamide gel electrophoresis of the 26S proteasomes isolated from various rpn10-complemented lines showed the correlation of the extent of reduced abundance of the double-capped 20S proteasomes with the severity of growth defects. In addition, mass spectrometry analyses of the 26S proteasomes isolated from rpn10 showed increased integration of specific subunit paralogues and increased association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. In the same report, extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and a UCH2-binding region in the RPN13 C-terminus (246–254). Interestingly, an analysis of homozygous rpn10-2 segregation in a rpn13-1 background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13–RPN2 association in vivo.