Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13

Background: The double-capped 26S proteasome complex is viewed as a symmetrical particle. Results: The ubiquitin receptor subunit Rpn13 is found in an asymmetrical ratio. Conclusion: Rpn13 ubiquitin receptor may dictate preference in substrate recruitment and processing. Significance: The ubiquitin receptor Rpn13 defines asymmetry and thus imposes directionality in the 26S proteasome. The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.