It is widely accepted that stress granules (SGs) are composed of stalled translation preinitiation complexes (48S) containing mRNA, small ribosomal subunits and some translation initiation factors 
. Here we provide evidence that the heat-induced SGs formed at 46°C in S. cerevisiae
also contain certain translation elongation and translation termination factors. Some of them, elongation factors eEF3 and eEF1Bγ2 and the termination factor eRF1 accumulate already at 42°C on Dcp2 foci independently of P-bodies scaffolding proteins. We also showed, on the example of eEF3 factor, that an accumulation of the protein at 42°C is independent on the Gcn2 kinase activity. However, these foci still determine sites for assembly of SGs upon heat shock at 46°C. Our data suggest that assembly of these SGs might be controlled by translation elongation and termination factors released from ongoing translation. Furthermore, recruitment of the key translation initiation factor eIF2α (Sui2p) to dissolving SGs points to the recovery of translation at these sites after stress relief.
Although SGs are referred to as stalled translation preinitiation complexes, it is generally accepted that the composition of SGs varies depending on organisms and cell types, as well as on intensity and type of the stress 
. For example, SGs induced by a high concentration of ethanol in S. cerevisiae
contain only the eIF3c/Nip1 subunit of the eIF3 complex 
and SGs induced by a prolonged glucose deprivation do not harbor the eIF3 complex at all 
. With respect to the intensity of the stress, treatment with a low concentration of NaN3
does not affect the distribution of eIF3a (Rpg1p/Tif32p) 
but at higher concentration this drug induces eIF3a accumulation in SGs 
. We show here that SGs induced by the robust heat shock in S. cerevisiae
contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) together with translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). These factors have never been observed in SGs of any other eukaryotic cell. However, the termination factors have been found to accumulate in P-bodies 
. Those authors have concluded that presence of translation termination factors in P-bodies is coupled to the P-bodies assembly. A similar role could be suggested for presence of these factors in heat-induced SGs.
The proteins with self-aggregation (prion-like) domain, like TIA-1 or TIAR in mammalian cells, have been described to influence dynamics of SGs 
. A newly identified component of the heat-induced SGs in S.cerevisiae
, Sup35p, possesses a prion-like domain at the N-terminus. Sup35p can thus convert into the prion form, known as [PSI+]. The N-terminal part of the protein is indispensable for the prion formation and maintenance 
. Similarly to a situation in mammalian cells 
we found that rather a non-prion part of Sup35p is responsible for accumulation of the protein in SGs. However, observations that SGs are formed even in the absence of the N-terminal prion-like domain of Sup35p indicate that unlike in mammals, the assembly of heat-induced SGs in S. cerevisiae
is not driven by these “prion” structural elements. This hypothesis is also supported by our earlier findings that heat-induced SGs are formed even in the absence of yeast orthologs of mammalian TIA-1 and TIAR proteins, Ngr1 and Pub1 proteins in S. cerevisiae
Translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p) are responsible for effective termination of translation 
. In addition, they seem to be required for an effective function of the fungal-specific elongation factor eEF3 (Yef3p) 
in recycling of the translation posttermination complexes after the release of newly synthetized peptide chains 
. In this respect, identification of elongation and termination factors in heat-induced SGs may indicate that these SGs are composed of translation posttermination complexes stalled before the ribosome recycling step. However, we did not observe any accumulation of several essential proteins of the 60S ribosomal subunits under robust heat shock 
and Grousl et al. (unpublished data). In addition, all the published information on recycling of the translation posttermination complexes comes from in vitro experiments only. Therefore, it is currently unclear, how recycling is catalyzed in vivo and the reasons for presence of the translation elongation and the termination factors in robust heat shock-induced SGs remain to be elucidated.
Whereas different roles for SGs and P-bodies in cell survival upon heat stress conditions could be suggested, both accumulations are always closely spatially and functionally intertwined. In S. cerevisiae
cells, P-bodies promote formation of SGs 
. The assembly of P-bodies is connected with changes in expression profiles and adaptation to changed environmental conditions, when the translation of certain transcripts is inhibited and, on the other hand, the translation of new transcripts is induced 
. They are present in cells even under non-stress conditions and they enlarge under various stresses, such as a heat shock at 39°C 
and 42°C 
. However, one of the major components of P-bodies, Dcp2p that is engaged in mRNA decapping 
, is also a component of the robust heat shock-induced SGs, which may form independently of the P-bodies scaffolding proteins Edc3 and Lsm4 
. On the contrary to glucose-deprived cells 
and cells heat-shocked at 37°C, we show here that Dcp2 foci formed in cells heat-shocked at 42°C do not depend on these scaffolds. These Dcp2 accumulations do not contain translation initiation factors and still serve as sites for assembly of SGs upon continuous and more robust heat stress. They might be considered as “premature SGs”, as well as Dcp2-containing structures related to P-bodies.
The stress-induced phosphorylation of translation initiation factor eIF2α is the best characterized mechanism of stress granule assembly 
. However, there are other ways, how to induce SGs or influence their dynamics. Apart influencing other translation initiation factors, they concern the metabolism of polyamines or hexosamines and the stress-induced tRNA derivates 
. Moreover, since eIF2α-phosphorylation independent mechanisms of SGs assembly prevail in lower eukaryotes, it seems that these are evolutionary older. We show here that accumulations of translation elongation factor eEF3 (Yef3p) on Dcp2 foci at 42°C precede assembly of eIF3-containing SGs in cells heat-shocked at 46°C. This suggests that the translation elongation phase is affected first in the stressed cells. It is conceivable that there might be a shortage of the eEF3 factor due to its sequestration into cytoplasmic foci at 42°C. This may cause an alteration of kinetics of the translation elongation, which results in a deceleration of translation initiation. Such regulation of translation at the elongation step has also been proposed as a possible function of the Stm1 protein, which is able to stall ribosomes after the 80S complex formation in vitro
and to promote decapping of a subset of mRNA 
. Moreover, Stm1p has been shown to regulate interaction of eEF3 factor with ribosomes and to play a complementary role to eEF3 in translation under nutrient stress conditions 
. Interestingly, we did not observe an accumulation of Stm1-GFP fusion protein neither upon heat shock at 42°C nor under heat shock at 46°C (Figure S3
). Additionally, we did not see any effect of the stm1
Δ on assembly of SGs in cells heat-shocked at 46°C (Figure S4
). Therefore, the roles of Stm1 protein and eEF3 factor in the Gcn2-independent signaling and translation repression resulting in SGs assembly in heat-shocked cells remain elusive.
Meanwhile assembly of P-bodies is generally connected with reprogramming of cells to new growth conditions, SGs are formed in response to severe stresses when the translation of housekeeping genes is completely shut down. Although SGs are thought to be sites where mRNA molecules are sorted, selected, and together with translation factors, sheltered from the effects of a stress 
, the fate of SGs components after a stress relief is mainly unknown 
. However, it is conceivable that at least some SGs protein components may also return back to the active translation. In this respect, our observation of accumulation of the key translation initiation factor eIF2α (Sui2p) on dissolving SGs during cell recovery from the heat stress suggests that SGs may help cells to effectively recover after a stress relief. To recover, a fast restart of translation is facilitated by increasing a local concentration of translation initiation and elongation components in SGs, where only the key regulator, eIF2α factor (Sui2p), is missing and recruited after a stress relief only. There is a supporting evidence from mammalian cells where a phospho-variant of the eIF2α factor subunit was found to be recruited to disassembling SGs and considered as important for SGs disassembly 
. On the contrary, we found that the eIF2α factor (Sui2p) accumulation on SGs does not depend on the phosphorylation status of this factor. It implies that the eIF2α factor is recruited to dissolving SGs also in its unphosphorylated state, thus translation competent. Taken together, it reinforces the hypothesis that SGs serve as sites where translation is effectively initiated at the time of a stress relief.
We showed here that a portion of the key translation initiation factor eIF2α (Sui2p) is recruited to dissolving SGs, but some of the Sui2-GFP foci did not co-localize with SGs markers in these cells. We suggest that these particular Sui2-GFP foci may represent the eIF2B bodies 
. In accordance with our assumption that translation is restored on dissolving SGs, the eIF2B bodies should also be formed under recovery from the stress. The eIF2B bodies most probably serve as sites, where guanine nucleotide exchange of the eIF2α factor takes place and the eIF2α-GDP form is converted to the translation competent eIF2α-GTP form. The eIF2B bodies would then help to regenerate efficiently the translation competent form of the eIF2α factor as suggested previously 
. The eIF2α-GTP form would then be recruited to sites on dissolving SGs.
Altogether, our data support the current view that the composition of stress granules depends on the type and the intensity of the applied stress. We confirmed that formation of yeast heat shock-induced SGs is not dependent on the translation initiation arrest caused by phosphorylation of eIF2α and we propose that translation machinery in heat shocked-cells seems to be primarily modulated at the level of translation elongation since also some translation elongation and termination factors accumulate within SGs. Our data further indicate that SGs reflect the sites where translation initiates after a stress relief. We also show that RNP accumulations formed upon heat shock at 42°C and containing translation elongation and termination factors may develop into genuine SGs upon robust heat shock at 46°C. Although we confirmed that all these accumulations depend on mRNA released from translation, links between heat-induced repression of translation and SGs assembly still remain to be elucidated.