Abstract
Stress granule (SG) formation is frequently accompanied by alterations of the ubiquitin proteasome system (UPS) and accumulation of ubiquitylated proteins. Ubiquitin and UPS components have been reported to co-localize with SGs, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. Using ubiquitin-specific proteomics we show that ~35% of ubiquitylated peptides change significantly in abundance in response to sodium arsenite treatment and that chemical inhibition of ubiquitin-activating enzyme (UAE) results in the rapid near-complete loss of ubiquitylation regardless of chain type. Critically, neither inhibition of UAE, nor the NEDD8-activating enzyme (NAE), significantly affected SG formation or disassembly, indicating that neither active protein ubiquitylation nor neddylation is required for SG dynamics. Using antibodies with varying preference for free vs. poly-ubiquitin in combination with UAE inhibition, we show that SGs accumulate primarily unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions.
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