Ubiquitination is essential for recovery of cellular activities after heat shock

INTRODUCTION In response to many types of stress, eukaryotic cells initiate an adaptive and reversible response that includes down-regulation of key cellular activities along with sequestration of cytoplasmic mRNAs into structures called stress granules. Accompanying these stress responses is a global increase in ubiquitination that has been conventionally ascribed to the need for degradation of misfolded or damaged proteins. However, detailed characterization of how the ubiquitinome is reshaped in response to stress is lacking. Furthermore, it is unclear whether stress-dependent ubiquitination plays a more complex role in the larger stress response beyond its known protective function in targeting hazardous proteins for proteasomal degradation. RATIONALE To explore the role of ubiquitination in the stress response, we used tandem ubiquitin binding entity (TUBE) proteomics to investigate changes to the ubiquitination landscape in response to five different types of stress in cultured mammalian cells, including human induced pluripotent stem cell (iPSC)–derived neurons. The discovery of unanticipated patterns of ubiquitination prompted a detailed analysis of the ubiquitination pattern specifically induced by heat shock by using diGly ubiquitin remnant profiling along with tandem mass tag quantitative proteomics in combination with additional total proteome and transcriptome analyses. Insights from this newly defined “heat shock ubiquitinome” guided subsequent investigation of the functional importance of this posttranslational modification in the cellular response to heat shock. RESULTS Each of the five different types of stress induced a distinctive pattern of ubiquitination. The heat shock ubiquitinome in human embryonic kidney 293T cells was defined by ubiquitination of specific proteins that function within cellular activities that are down-regulated during stress (e.g., translation and nucleocytoplasmic transport), and this pattern was similar in U2OS cells, primary mouse neurons, and human iPSC-derived neurons. The heat shock ubiquitinome was also enriched in protein constituents of stress granules. Suprisingly, this stress-induced ubiquitination was dispensable for the formation of stress granules and shutdown of cellular pathways; rather, heat shock–induced ubiquitination was a prerequisite for p97/valosin-containing protein (VCP)–mediated stress granule disassembly and for resumption of normal cellular activities, including nucleocytoplasmic transport and translation, upon recovery from stress. Many ubiquitination events were specific to one or another stress. For example, ubiquitination was required for disassembly of stress granules induced by heat stress but dispensable for disassembly for stress granules induced by oxidative (arsenite) stress. CONCLUSION Ubiquitination patterns are specific to different types of stress and indicate additional regulatory functions for stress-induced ubiquitination beyond the removal of misfolded or damaged proteins. Specifically, heat shock–induced ubiquitination primes the cell for recovery from stress by targeting specific proteins involved several pathways down-regulated during stress. Furthermore, some key stress granule constituents are ubiquitinated in response to heat stress but not arsenite stress, thus engaging a mechanism of VCP mediated–disassembly of heat shock–induced granules that is not shared by arsenite stress–induced granules. Finally, our deep proteomics datasets provide a rich community resource illuminating additional aspects of the roles of ubiquitination in response to stress.