STI1 domains coordinate partitioning of UBQLN2 into stress-induced condensates

Abstract

UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation in vitro and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that in vitro phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions. HighlightsO_LIUBQLN2 is recruited to both stress granules and puncta formed by the autophagy receptor protein p62 in response to heat stress. C_LIO_LIBoth endogenous and overexpressed UBQLN2 tend to colocalize with p62 puncta in cells where p62 is abundant, even without acute stress treatment. C_LIO_LIUsing an extensive domain deletion library, ability of UBQLN2 constructs to form puncta correlates with their in vitro phase separation propensity in the absence of stress. C_LIO_LIEach of the two STI1 domains contribute non-redundantly to formation of UBQLN2 puncta in response to heat stress. C_LIO_LIThe STI1-II domain is independently required for UBQLN2 oligomerization, phase separation, and puncta formation. C_LI