Novel Components of the Stress Assembly Sec Body Identified by Proximity Labeling.

Sec bodies are membraneless stress-induced assemblies that form by the coalescence of endoplasmic reticulum exit sites (ERES). Through APEX2 tagging of Sec24AB, we biotinylated and identified the full complement of Sec body proteins. In the presence of biotin-phenol and H2O2 (APEX on), APEX2 facilitates the transfer of a biotin moiety to nearby interactors of chimeric Sec24AB. Using this unbiased approach comparing APEX on and off (-H2O2) conditions, we identified 52 proteins specifically enriched in Sec bodies. These include a large proportion of ER and Golgi proteins, packaged without defined stoichiometry, which we could selectively verify by imaging. Interestingly, Sec body components are neither transcriptionally nor translationally regulated under the conditions that induce Sec body formation, suggesting that incorporation of these proteins into granules may be driven instead by the aggregation of nucleating proteins with a high content of intrinsically disordered regions. This reinforces the notion that Sec bodies may act as storage for ERES, ER and Golgi components during stress.

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper.

The function of GORASPs in Golgi apparatus organization in vivo.

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

Phospho-Rasputin Stabilization by Sec16 Is Required for Stress Granule Formation upon Amino Acid Starvation.

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein.