Molecular basis for yeast ubiquilin Dsk2 self-association and phase separation

Ubiquilins are a group of highly conserved genes found in various organisms, including yeast, humans, and plants. They play a crucial role in protein quality control mechanisms, including the ubiquitin-proteasome system and autophagy. Ubiquilins have a multi-domain architecture generally consisting of three main domains: an N-terminal ubiquitin-like (UBL) domain, a C-terminal ubiquitin-associated (UBA) domain, and a variable number of internal STI1 repeats. The UBL domain interacts with the proteasome and proteins containing ubiquitin-interacting motifs, while the UBA domain helps in binding to polyubiquitinated proteins. While the function of UBL and UBA regions of ubiquilins are well-known, the structure and functional role of the STI1 is poorly understood. We recently showed that the central regions (STI1s) are important for UBQLN self-association and phase separation. In contrast, for the yeast ubiquilin homologue, Dsk2, it has been observed that the UBL and UBA domains can interact with each other, leading to self-association. However, the specific function of STI1 in the functioning of Dsk2 remains to be explored. In our current study, we investigate how the STI1 regions and other disordered regions of Dsk2 are involved in its self-association, phase separation, and binding to ubiquitinated substrates. We show that Dsk2 phase separates in the presence of crowding agents. We employ biomolecular NMR techniques in conjugation with other biophysical tools to map Dsk2 self-association behavior at an atomic level.