Processing body and stress granule assembly occur by independent and differentially regulated pathways in Saccharomyces cerevisiae

Abstract

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.

Figure 1

The presence of Ras/PKA signaling activity was sufficient to trigger P body disassembly. (A) The ectopic expression of the constitutively active Ras2^val19 protein resulted in the dispersal of Edc3-containing foci. Cells expressing the Edc3-mCherry protein were transferred to a glucose-minus medium containing 250 mM methionine for 10 min to induce P body formation. The cells were then moved to a medium that also lacked methionine for 2 hr to induce expression from the MET3-RAS2^val19 locus and then were examined by fluorescence microscopy. Three representative images are shown for both the control and the induced conditions. (B) Increased Ras2 protein expression was detected in cells containing the MET3-RAS2^val19 locus. Protein extracts were prepared from cells containing either a control vector or a MET3-RAS2^val19 plasmid, and the level of Ras2 protein was assessed by Western blotting with an anti-Ras2 antibody. (C and D) The RAS2^val19-triggered disassembly of P bodies was blocked in cells containing the nonphosphorylatable variant Pat1-AA. The disassembly of preformed P bodies was assessed as described above in cells expressing GFP-tagged versions of either the wild-type Pat1-SS or the nonphosphorylatable Pat1-AA. The microscopy data are shown in C, and the quantitation of these data in D. The data shown in the graph represent the average of at least two experiments (n = 100) with the Edc3-mCh reporter.

Figure 2

All three isoforms of the S. cerevisiae PKA were able to influence P body formation. (A and B) The overexpression of any one of the three Tpk proteins was sufficient to inhibit P body formation. Cells containing either the control vector or a plasmid with the indicated TPK gene were transferred to a glucose-minus medium for 10 min to induce P body formation. The cells also expressed either the Pat1-SS or Pat1-AA protein, as indicated. The microscopy data are presented in A and the quantitation of these data in B. (C) The PKA site in Pat1 was phosphorylated in cells lacking any two of the three Tpk isoforms. Pat1 was immunoprecipitated from log-phase cells with the indicated genotypes, and the level of in vivo PKA phosphorylation was assessed by Western blotting with an antibody that specifically recognizes the phosphorylated form of Pat1 (α-Sub). (Top) Western blot showing the total amount of the myc-tagged Pat1 present. (D) Pat1 was phosphorylated in vitro by the Tpk1 and Tpk2 isoforms of PKA. The myc-tagged Pat1 proteins were immunoprecipitated from log-phase cells, treated with phosphatase, and then incubated with the indicated GST-tagged Tpk protein and [γ-³²P]ATP. The bottom two panels are Western blots indicating the total amount of the respective proteins present in the reaction mixtures. “(-)” indicates the “no kinase” control. (E) P body formation in mutants containing only a single Tpk isoform. Cells with the indicated genotypes were transferred to a medium lacking glucose for 10 min, and P body formation was then assessed by fluorescence microscopy.

Figure 3

Ras/PKA signaling activity inhibited P body formation in response to hyperosmotic stress. (A) Osmotic stress-induced P body formation was inhibited in cells expressing the constitutively active Ras2^val19 protein. Cells transformed with either a control vector or a plasmid containing the RAS2^val19 locus were grown to mid-log phase and transferred to a medium either lacking glucose (−Glu) or containing 1 M KCl for 10 min. P body formation for each of the indicated reporters was then assessed by fluorescence microscopy. (B) The PKA-mediated inhibition of P body formation was suppressed by the presence of the nonphosphorylatable Pat1-AA. P body formation in response to 1 M KCl was assessed as above for pat1Δ cells expressing GFP-tagged versions of either Pat1-SS or Pat1-AA. (C) Quantification of the data shown in B. The graph indicates the fraction of cells with Pat1-containing foci after a 10-min exposure to 1 M KCl. (D) The PKA-dependent phosphorylation on Pat1 was rapidly diminished upon exposure to 1 M KCl. Cells expressing a myc-tagged Pat1 were grown to mid-log phase and then transferred for 10 min to the same medium (SC glucose; +Glu) or media either lacking glucose (−Glu) or containing 1 M KCl. The level of PKA phosphorylation was assessed by Western blotting with the anti-PKA substrate antibody (α-Sub). Untr, the extract prepared from the untreated control culture. (E) The frequency of P body formation following an osmotic stress was diminished in cells lacking Pat1. Wild-type and pat1Δ cells expressing the Edc3-mCh reporter were exposed to a medium containing 1 M KCl for 10 min and then examined by fluorescence microscopy.

Figure 4

The presence of elevated PKA signaling activity inhibited P body induction in response to a variety of stress conditions. (A and B) Expression from the RAS2^val19 locus inhibited the P body formation brought on by a number of stress conditions. Wild-type cells expressing the Edc3-mCh reporter were transformed with either a RAS2^val19 plasmid or a control vector and exposed to the indicated stress for 10 min. P body formation was then assessed by fluorescence microscopy. The microscopy data are presented in A and the quantitation of these data in B. The 1 M KCl microscopy data are shown in Figure 3A. (C) The presence of the nonphosphorylatable Pat1-AA diminished the inhibitory effects of the Ras2^val19 protein. P body foci formation was assessed after a 10-min exposure to the indicated stress in pat1Δ cells expressing GFP-tagged Pat1-SS or Pat1-AA. The cells contained either a control vector or a RAS2^val19 plasmid, as indicated. A graph showing the quantitation of these data are presented in Figure S2A. (D) P body formation in response to a variety of stresses required the presence of the Pat1 protein. P body formation was assessed in wild-type and pat1Δ cells after a 10-min exposure to the indicated conditions. A graph showing the quantitation of these data is presented in Figure S2B.

Figure 5

Stress granule formation was not influenced by Ras/PKA signaling activity. (A and B) Elevated Ras/PKA activity did not inhibit stress granule formation in response to a number of inducing conditions. Wild-type cells expressing either Pab1-GFP (A) or Pbp1-GFP (B) were transformed with either a control vector or a RAS2^val19 plasmid. Stress granule formation was induced by a 30-min exposure to a 46° heat stress (Heat) or media either lacking glucose or containing 15% ethanol or 0.5% NaN₃, as indicated. (C) The down-regulation of PKA signaling did not result in stress granule formation. Plasmids with GFP-tagged versions of either Pab1 or Pat1 were introduced into a yeast strain (PHY4697) with an analog-sensitive version of TPK1, tpk1^as. The cells were then treated for 30 min with 5 μM 1NM-PP1 to inhibit the encoded Tpk1^as protein, and foci formation was assessed by fluorescence microscopy. (D) Stress granule formation was not induced upon inactivation of the TORC1 signalling pathway. Wild-type cells expressing the Pab1-GFP fusion protein were treated for 20 min with 200 ng/ml rapamycin. (E) Stress granule formation in pat1Δ cells. Wild-type and pat1Δ cells were exposed to the indicated stresses for 30 min, and Pab1-GFP foci were visualized by fluorescence microscopy.

Figure 6

The presence of the phosphomimetic Pat1-EE resulted in fewer P body foci and a reduced survival in stationary phase. (A and B) Cells with the Pat1-EE variant had fewer P body foci. The number of P body foci after 5 days of growth in YPAD medium was assessed for cells expressing GFP-tagged versions of either Pat1-SS or Pat1-EE. Three representative images are shown for cells containing either Pat1-SS or Pat1-EE. The microscopy data are shown in A and the quantitation of these data in B. (C) Cells expressing the Pat1-EE variant had a diminished CLS. The number of viable cells present during log-phase growth and after 10 days in a minimal YM glucose medium was determined by plating out increasing dilutions of these cultures. (D) Deletion of the PAT1 locus suppressed the extended CLS of a ras2Δ mutant. Serial dilutions of strains with the indicated genotypes were plated out at the indicated times of culture growth in a YM glucose minimal medium.

Figure 7

P body foci are present and persist throughout the stationary phase of growth. (A) P body foci are present in stationary-phase cells. The frequency of P body formation was determined for cells with the integrated Dcp2-GFP and Dhh1-GFP reporters at the indicated times of growth in YPAD cultures. (B) A graph showing the number of Dcp2 foci at the indicated culture times; the relevant microscopy data are presented in A. (C and D) The presence of the constitutively active Ras2^val19 protein resulted in fewer P body foci. P body foci were visualized by fluorescence microscopy after the indicated number of days of growth for wild-type cells with either a control vector or a RAS2^val19 plasmid. (E) A graph showing the quantitation of the Dcp2-GFP microscopy data presented in D. (F and G) The presence of the Pat1 protein was required for the formation of stationary-phase P body foci. The fraction of cells with P body foci was determined for wild-type and pat1Δ cells after the indicated number of days of growth in YPAD. The results for a 10-min starvation for glucose are shown for comparison purposes (−Glu). The microscopy data are shown in F and the quantitation of these data in G.

Figure 8

Stress granules appeared in cells after entry into stationary phase. (A) A representative growth curve for a wild-type strain with the integrated Pbp1-GFP reporter. The red lines delimit the log, PDS, and stationary phases of growth. (B) Stress granule foci were present in stationary-phase cells. The subcellular localization of Pab1-GFP and Pbp1-GFP was assessed by fluorescence microscopy after the indicated days of growth in the rich growth medium YPAD. (C) Stress granule formation was not affected by the presence of the RAS2^val19 locus. The subcellular localization of a Pbp1-GFP reporter was assessed after the indicated number of days of growth for cells containing either a control vector or a RAS2^val19 plasmid. (D and E) Stress granule foci did not appear in significant numbers until after cells had entered into the stationary phase of growth. The subcellular localization of the indicated reporters was assessed in cells at daily intervals of growth in a rich medium. The graph in E shows the fraction of cells with a Pbp1-GFP focus after the indicated days of growth. (F) Pbp1 was found in the nucleus during the PDS period of growth. Cells expressing Pbp1-GFP were transformed with a plasmid encoding the nuclear marker, histone H2B-mCh, and the colocalization of the two reporters was assessed after 3 days of culture growth.

Figure 9

Mutants lacking the stress granule protein Pub1 have a diminished CLS and fewer stationary-phase P body foci. (A) pub1Δ mutants exhibited a decreased rate of survival during stationary phase. The number of viable cells present at the indicated times of growth was determined by plating out increasing dilutions of the cultures as described in Materials and Methods. (B and C) The subcellular localization of a stress granule (B: Pab1-GFP) or P body (C: Edc3-mCh) marker was assessed by fluorescence microscopy after the indicated days of growth. (D) Quantitation of the P body foci data for the wild-type and pub1Δ strains shown in C.