2078 Research Article Robust heat shock induces eIF2α-phosphorylation- independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae Tomás Grousl1, Pavel Ivanov1,*, Ivana Frydlová1, Pavla Vasicová1, Filip Janda1, Jana Vojtová1, Katerina Malínská1, Ivana Malcová1, Lenka Nováková1, Dana Janosková1, Leos Valásek2 and Jirí Hasek1,‡ 1Laboratory of Cell Reproduction, Institute of Microbiology of the AS CR, v.v.i., Prague, Czech Republic 2Laboratory of Regulation of Gene Expression, Institute of Microbiology of the AS CR, v.v.i., Prague, Czech Republic *Present address: A.N. Belozersky Institute of Physico-Chemical Biology MSU, Moscow, Russia ‡Author for correspondence (e-mail: [email protected]) Accepted 18 March 2009 Journal of Cell Science 122, 2078-2088 Published by The Company of Biologists 2009 doi:10.1242/jcs.045104 Summary Environmental stresses inducing translation arrest are markers also colocalized with eIF3a. Microscopic analyses of accompanied by the deposition of translational components into the edc3Δlsm4ΔC mutant demonstrated that different stress granules (SGs) serving as mRNA triage sites. It has scaffolding proteins are required to induce SGs upon robust recently been reported that, in Saccharomyces cerevisiae, heat shock as opposed to glucose deprivation. Even though formation of SGs occurs as a result of a prolonged glucose eIF2α became phosphorylated under these stress conditions, the starvation. However, these SGs did not contain eIF3, one of decrease in polysomes and formation of SGs occurred hallmarks of mammalian SGs. We have analyzed the effect of independently of phosphorylation of eIF2α. We conclude that robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and under specific stress conditions, such as robust heat shock, yeast showed that it results in the formation of eIF3a accumulations SGs do contain eIF3 and 40S ribosomes and utilize alternative containing other eIF3 subunits, known yeast SG components routes for their assembly. and small but not large ribosomal subunits and eIF2α/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body Key words: P-bodies, Stress granules, Yeast Journal of Cell Science Introduction P-bodies were originally described as sites of mRNA decapping Intracellular compartmentalization of specific mRNAs and and decay, which mainly contain the mRNA degradation components of the translation machinery is an important mode of machinery components including the decapping enzyme complex regulation for gene expression in eukaryotic cells. In this respect, (Brengues et al., 2005). P-bodies and SGs share some proteins formation of various mRNA-containing assemblies, such as stress and mRNA components, but also contain a number of unique granules (SGs) or processing bodies (P-bodies), is a striking markers specific to each structure (Kedersha and Anderson, illustration of this regulation (Anderson and Kedersha, 2008; Bond, 2007). Unlike SGs, P-bodies are present even under non-stress 2006; Parker and Sheth, 2007). conditions (Mollet et al., 2008) and never contain eIF3 and 40S Various stresses cause a fast and transient redistribution of ribosomal subunits (Anderson and Kedersha, 2008; Brengues and nontranslated mRNAs into SGs in mammalian as well as plant cells. Parker, 2007; Parker and Sheth, 2007; Sheth and Parker, 2003; This effect is a result of a rapid repression of general translation Sheth and Parker, 2006). P-bodies are therefore believed to be initiation, often mediated through phosphorylation of the alpha structurally distinct from SGs; however, their simultaneous subunit of the translation initiation factor 2 (eIF2) (Holcik and appearance in stressed mammalian cells showed a close spatial Sonenberg, 2005; Kedersha et al., 1999). Therefore mammalian SGs connection perhaps indicating an intimate mutual communication are thought to represent abortive 48S complexes that include mRNA (Kedersha et al., 2005; Wilczynska et al., 2005). In addition, linked to poly(A)-binding protein 1, translation initiation factors because of these interactions, a role for pre-existing P-bodies in (e.g. eIF3, eIF4A, eIF4G) and 40S ribosomal subunits (Anderson the nucleation of SG assembly has been suggested recently and Kedersha, 2008; Kedersha et al., 2002). SGs persist in (Buchan et al., 2008; Mollet et al., 2008). equilibrium with polysomes and this can be affected by translation In the budding yeast S. cerevisiae, a number of small cytosolic inhibitors, which either stabilize or destabilize the polysomes. It foci of accumulated Dcp2p can be observed also in normal has been proposed that the SGs serve as triage sites redirecting proliferating cells (Brengues et al., 2005; Teixeira et al., 2005). mRNA to either translation, storage or degradation (Anderson and Under various stresses, e.g. glucose starvation, hyperosmotic stress Kedersha, 2008). Interestingly, SG-like structures, containing eIF3, and heat stress at 37°C, these foci increase in size to form enlarged have been found in heat-shocked fission yeast Schizosacchomyces P-bodies (Brengues et al., 2005). Recently, novel mRNA assemblies pombe (Dunand-Sauthier et al., 2002), but have not yet been reported called EGP-bodies (for eIF4E, eIF4G and Pab1p) that are distinct in Saccharomyces cerevisiae. from P-bodies were found to form under prolonged glucose Yeast stress granules containing eIF3 2079 starvation in S. cerevisiae (Hoyle et al., 2007). While this eIF3 complex. As confirmed by high values of the Pearson’s manuscript was under a review process in this journal, additional correlation coefficient (Rr), over 0.9 (see Materials and Methods), markers of mammalian SGs were identified within EGP-bodies distribution for both eIF3b and eIF3c signals almost completely that led the authors to rename them ‘yeast stress granules’ (Buchan matched that of eIF3a. et al., 2008). In sharp contrast to mammalian SGs, however, the In living yeast, accumulation of mRNA in P-bodies has been reported yeast SGs of glucose-deprived yeast cells do not contain assessed using a specific PGK1 reporter mRNA containing multiple eIF3 and 40S ribosomal subunits (Buchan et al., 2008; Hoyle et U1A-specific binding sites in its 3Ј untranslated region to which U1A- al., 2007). GFP fusion protein binds (Brengues et al., 2005; Teixeira et al., 2005). In S. cerevisiae, numerous stresses including hyperosmolarity, We employed this detection system in order to find out if mRNA glucose deprivation and robust heat shock at 46°C were also accumulates in the eIF3a foci after robust heat shock. As shown demonstrated to stabilize many mRNAs (Hilgers et al., 2006). in Fig. 1E, the PGK1 mRNA in unstressed cells was distributed across Whereas the impact of hyperosmolarity on formation of P-bodies the cytosol, as has previously been observed (Brengues et al., 2005). seems to be identical to that of glucose deprivation (Brengues et In cells heat-shocked in a complete medium at 46°C for 10 minutes, al., 2005), it is not known whether robust heat shock also elicits the cytoplasmic GFP signal corresponding to the accumulated PGK1 similar effects. In this report, we show that robust heat shock at mRNA significantly overlapped with that of eIF3a foci. Weak 46°C for 10 minutes applied to aerobically cultivated yeast cells background fluorescence was observed in the cytoplasm of control results in formation of accumulations containing eIF3 subunits, cells expressing the U1A-GFP fusion protein only, without PGK1 mRNA, eIF4G2, Pab1p, Ngr1p, Pub1p, 40S ribosomal subunits and mRNA under all tested conditions (data not shown). also typical P-body proteins such as Dcp2p and Dhh1p. Assembly of these transient protein accumulations is eIF2α-phosphorylation eIF3a foci contain typical components of the mammalian SGs independent. We also demonstrate that they are not formed in Stalled 48S pre-initiation complexes containing mRNA, eIF3, cycloheximide-treated and energy-depleted cells. Because of their eIF4G, PABP and 40S, but not 60S ribosomal subunits and eIF2, composition, irregular shapes and requirement for different are the core constituents of the mammalian SGs (Kedersha et al., scaffolding proteins than are those needed for P-bodies, we posit 2002). To determine whether yeast eIF3 foci are related to that these protein accumulations are yeast SGs specific for the robust mammalian SGs, we constructed strains coexpressing various RFP heat shock. We further propose that they can only be detected in and GFP fusion proteins, exposed the exponentially growing cells S. cerevisiae when transient severe stresses such as robust heat shock to robust heat shock in YPD medium at 46°C for 10 minutes and are applied and the period of yeast mRNA triage is relatively analyzed the distribution of fusion proteins using the Olympus Cell prolonged. R microscopic system. First, we found that in the heat-shocked cells eIF3a colocalized Results with eIF4G2 and Pab1p (Fig. 2A). Very high values of Pearson’s Robust heat shock induces transient formation of eIF3- correlation coefficient (Rr), over 0.8, confirmed a high degree of containing SGs colocalization. In addition, we also analyzed changes in the We showed previously that eIF3a/Tif32p/Rpg1p colocalized with distribution of Rps30A-GFP fusion protein that we used as a marker Journal of Cell Science cytoplasmic microtubules in the fixed yeast cells (Hasek et al., of the 40S ribosomal subunit. We found that robust heat shock 2000). In order to examine intracellular distribution of eIF3a