Precise Mapping of the Proteasome Interaction Region (PIR) of p62/SQSTM1: Decoupling Condensate Formation from Proteasome Recruitment.

p62/SQSTM1 is a multifunctional scaffold protein central to selective autophagy and, more recently, recognized as a regulator of ubiquitin-proteasome system-mediated degradation of intracellular proteins. Within phase-separated condensates, p62 has been shown to recruit and sequester the proteasome, yet the molecular basis for this interaction has remained largely unknown. Our previous study demonstrated that the 'PB1' domain (residues 1-123) of p62 is necessary for proteasome binding. However, this long stretch is also responsible for other functions of p62, such as condensate assembly and signal transduction. Thus, it was important to define more precisely the region responsible for interaction with the proteasome. In this study, we used systematic deletion variants of p62 and biochemical assays to delineate the minimal sequence within the PB1 domain responsible for proteasome binding. Our analyses revealed a small stretch of six amino acids (residues 84-89) that bind the proteasome and are distinct from the region responsible for condensate formation. Such a precise variant can serve as a useful tool to dissect how p62-proteasome interaction affects selective degradation and probably stress response, separating it from other p62 functions. Overall, this work advances our understanding of the structural determinants underlying p62's dual role in autophagy and UPS regulation.