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

Abstract

SUMMARY 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.

Rasputin a decade on and more promiscuous than ever? A review of G3BPs

“…Under normal conditions, G3BPs are phosphorylated and in some reported cases, this causes mRNA degradation, whereas upon cellular stress induced by arsenite, unphosphorylated G3BPs may oligomerize and bring mRNAs to the SGs. In concordance with this, arsenite leads to unphosphorylation of G3BPs at Ser149 with subsequent SGs formation both in mammalian cells [14,86] and in Drosophila cells [87]. Whereas, upon the stress induced by amino acid starvation in Drosophila S2 cells, only the Ser142 phosphorylated Rasputin (G3BP) is recruited to SGs [87].…”

“…In concordance with this, arsenite leads to unphosphorylation of G3BPs at Ser149 with subsequent SGs formation both in mammalian cells [14,86] and in Drosophila cells [87]. Whereas, upon the stress induced by amino acid starvation in Drosophila S2 cells, only the Ser142 phosphorylated Rasputin (G3BP) is recruited to SGs [87]. Furthermore, Sec16 which is a component of endoplasmic reticulum (ER) exit site, interacts with and stabilizes phosphorylated Rasputin.…”

“…Furthermore, Sec16 which is a component of endoplasmic reticulum (ER) exit site, interacts with and stabilizes phosphorylated Rasputin. However, in absence of Sec16, the stabilization of phosphorylated Rasputin is not sufficient to form SGs, suggesting a critical role of Sec16 in SGs assembly upon amino acid starvation [87]. These studies suggest that SGs formation is a fine-tuned process which is regulated by specific signals that are unique to each stress.…”

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells

“…For instance, glucose starvation of yeast induces the formation of P‐bodies and of stress granules that appear to largely overlap with P‐bodies . Similarly, bona fide stress granule formation is induced by chronic or acute amino acid starvation in mammalian and Drosophila cells . However, whereas many stresses appear to solely lead to P‐bodies and stress granule formation, starvation appears to be a strong stress that leads to the formation of many different cytoplasmic stress assemblies, not all of which are RNA‐based (Figure ).…”

“…Tia‐1 (and TiaR) and G3BP1/2 are the two best characterized drivers for stress granule formation in vivo. Overexpression of either leads to ectopic stress granule formation even in the absence of stress, and their depletion prevents stress granule formation in both mammalian and Drosophila cells . A number of additional criteria establish that foci enriched in RNAs bound to RNA‐binding proteins are bona fide stress granules.…”