Widespread reorganization of proteins into cytoplasmic granules in stationary-phase yeast cells
A previous study has shown that many cytosolic proteins change their localization once yeast cells enter the stationary phase (Narayanaswamy et al., 2009
). To better understand whether epigenetic-related proteins are also under spatial regulation in quiescent cells, we monitored the subcellular localizations of 20 nuclear and cytoplasmic green fluorescent protein (GFP)-fusion proteins over a 4-wk time period (Huh et al., 2003
). These proteins comprised six histone deacetylases (Hda1, Hos2, Hst1, Hst2, Rpd3, and Sir2), two histone methyltransferases (Sds3 and Set2), four Sir2-interacting proteins (Net1, Rap1, Sir3, and Yku80), two nuclear pore complex–associated proteins (Nup49 and Mlp1), a histone protein (Htb1), an RNA polymerase II subunit (Rox3), a mitochondrial protein (Sod2), and three abundant cytosolic proteins (Sod1, Tdh2, and Yca1).
Most proteins we investigated changed their localization and formed cytoplasmic granules (SPGs), in quiescent cells. This result suggests that formation of cytoplasmic granules is a common behavior for proteins in quiescent yeast cells. These 20 proteins were divided into four groups according to their localization patterns during the progression of granule formation (summarized in Supplemental Table S1 and Figure S1). Different dynamics and morphologies between these granules suggest that they are probably distinct protein assemblies.
Hos2 proteins, but not all Set3/Hos2 complex components, relocalize into SPGs upon entry into quiescence
Previous studies have shown that the expression level of HOS
2 is upregulated in cells during diauxic shift, early stationary phase, and certain stresses (DeRisi et al., 1997
; Gasch et al., 2000
). We decided to further investigate the function of the Hos2 SPGs in stationary phase (a group I SPG in Supplemental Table S1). The histone deacetylase Hos2 is an essential component of the Set3/Hos2 complex, which is involved in meiotic gene repression and is also required for the activation of many genes (Pijnappel et al., 2001
; Wang et al., 2002
). In log-phase cells, Hos2 is diffused in the nucleus (, top panels). As cells entered stationary phase, usually one or a few bright granules were visible in the cytoplasm, with only small amounts of Hos2 still remaining in the nucleus (, bottom panels). The proportion of cells displaying normal nuclear diffusion of Hos2 dramatically decreased after 7 d of growth, and the percentage of cells with diffuse cytoplasmic patches gradually increased at later time points ().
FIGURE 1: Reorganization of Hos2 during stationary phase. (A) The deacetylase Hos2 is diffused in the nucleus in log-phase cells (top panels) but appears as a single bright Hos2 SPG in the cytosol upon entry into stationary phase (bottom panels). Cells were fixed (more ...)
Two experiments were conducted to ensure that our observations represented the behavior of wild-type Hos2 protein. First, we tested whether the Hos2-GFP protein could rescue the hypersensitivity of hos2
mutants to secretory stress when grown on a tunicamycin-containing plate (Cohen et al., 2008
). We observed that hos2
cells carrying the HOS2-GFP
construct were as resistant to tunicamycin as wild-type cells, indicating that the Hos2-GFP protein is functional. Next we fused the Hos2 protein with a 13×Myc tag and examined the protein localization using immunostaining. A similar localization pattern was observed, indicating that our previous data were not due to a GFP-specific artifact (Figure S2).
To determine whether other components of the Set3/Hos2 complex were relocalized to the same SPGs, stationary-phase cells coexpressing Hos2-mCherry with Cpr1-, Hos4-, Set3-, Sif2-, or Snt1-GFP fusion proteins were examined following growth in yeast–peptone–dextrose (YPD) medium for 1 wk (Figure S3). Both Set3-GFP and Sif2-GFP reorganized into SPGs upon entry into quiescence, and a proportion of these SPGs colocalized with Hos2 SPGs (61% of Set3-GFP dots and 74% of Sif2-GFP dots, n = 81–87). In contrast, Hos4-GFP, Snt1-GFP, and Cpr1-GFP formed dots occasionally but did not colocalize with Hos2 SPGs. We also examined the formation of Hos2 SPGs in set3
mutant cells in which the Set3/Hos2 complex is defective. Deletion of SET3
did not affect Hos2 SPG formation, suggesting that the assembly of Hos2 SPGs is distinct from the Set3/Hos2 complex.
A hos2 mutation affects cell viability and exit from stationary phase in quiescent cells
The Hos2 protein has been shown to function as a meiosis-specific repressor and an essential component of the secretory stress response (Pijnappel et al., 2001
; Cohen et al., 2008
). However, its role in cells during stationary phase is unknown. To address this question, we used in vivo time-lapse imaging to test whether a hos2
mutation would affect the survival or recovery of quiescent cells. Haploid wild-type and hos2
mutant cells were cultured in YPD medium for 4 wk. During this period, cells were collected at different time points and their recovery from stationary phase to log phase was examined (see Materials and Methods
for details). At the early time points (7 and 13 d), both wild-type and hos2
mutant cells maintained a high degree of viability. However, the cell viability in hos2
mutant cells was dramatically reduced after 24 d in YPD medium (). The loss of viability was not simply due to acidification of the medium, as a constant pH was maintained throughout the 4-wk period (Burtner et al., 2009
FIGURE 2: Hos2 is important for cell viability and growth recovery in stationary-phase cells. Stationary-phase cell cultures were diluted in fresh YPD, and cell growth was monitored using time-lapse microscopy. (A) hos2 mutant cells exhibited lower viability after (more ...)
We also analyzed the recovery process by determining the time required for cells to reenter the mitotic cell cycle after nutrient addition. In contrast to the viability assay, a significant difference between wild-type and hos2 mutant cells was detected in the 13-d samples (Mann-Whitney test, p < 0.01). The quiescent hos2 mutant cells took longer to reenter mitosis, and the delay increased as the cells aged (). We always observed two distribution patterns in the time of recovery, implying that the cell population was composed of two types of quiescent cells in stationary phase.
Hst2 and Yca1 colocalize with Hos2 SPGs during stationary phase
In our protein localization analyses, both Hst2-GFP and Yca1-GFP exhibited patterns similar to those of Hos2-GFP, suggesting they share similar characteristics during granule formation. This observation prompted us to investigate the possible association of Hos2 SPGs with either Hst2 or Yca1 protein. In 1-wk-old, nutrient-deprived cells containing Hos2 SPGs (1-wk cells), most Hos2 SPGs colocalized with Hst2-GFP (95 ± 5%, n = 194) and Yca1-GFP (91 ± 7%, n = 158; ). On the other hand, when we examined the Sir2 SPG, a cytoplasmic granule from another group (group II in Table S1), no significant colocalization with Hos2 SPGs or Yca1 SPGs was observed (Figure S4). These results suggest these proteins do not assemble by chance, and components of specific granules might be invariant.
FIGURE 3: Hos2 SPGs colocalize with Hst2 and Yca1, but not with actin bodies and proteasome storage granules. (A and B) Hos2-mCherry colocalizes with Hst2-GFP (95 ± 5%, n = 194) and Yca1-GFP (91 ± 7%, n = 158) in stationary-phase cells. (C) Actin (more ...)
Hos2 SPG in quiescent cells is a reversible structure
We next asked whether the SPGs in quiescent yeast cells are simply aggregates or specific, regulated structures. If the SPGs are formed by impaired or denatured proteins, it is unlikely that these proteins will be relocalized back to the nucleus when cells resume mitosis. On the other hand, a reversible granule suggests specific functions in quiescence. To clarify this issue, we monitored the localization of Hos2-GFP during the recovery process of 1-wk cells. After refeeding with nutrients, most Hos2 SPGs quickly dissolved, and the characteristic pattern of log-phase cells could be observed within 40 min (). Interestingly, no cells with Hos2 SPGs showed signs of rebudding, suggesting that disassembly of Hos2 SPGs may be crucial for cells to reenter mitosis.
FIGURE 4: Hos2 granules are reversible upon nutrient replenishment. (A) Time-lapse images of two recovering cells. Hos2-GFP–expressing cells were grown to stationary phase and then cultivated on a Lab-Tek chambered coverglass with fresh medium. Hos2 SPGs (more ...)
In 1-wk cells, more than 60% of cellular Hos2 protein was found in cytoplasmic SPGs. By contrast, Hos2 protein was nearly exclusively restricted to the nucleus after 2 h of replenishment with nutrients ( and S5). In addition, Hos2 SPGs could be detected in over 95% of 1-wk cells. After 2-h refeeding, the percentage of cells containing Hos2 SPGs was drastically reduced to ~20% (). In those cells that still contained Hos2 SPGs, the size and intensity of the granules were significantly decreased. We also measured the total amount of Hos2 in recovering cells. No obvious change was detected during the process of recovery, suggesting the Hos2 protein in SPGs was not degraded, but rather was redistributed from SPGs to the nucleus ().
When 2-wk cells were examined, we found that Hos2 SPGs took longer to disassemble in the recovering cells, which was consistent with our observation that older cells had a longer delay in reentering mitosis (). In the recovery experiments, we also monitored the other Hos2 SPG components, Hst2 and Yca1. Both proteins showed patterns similar to those of Hos2 (unpublished data).
Protein synthesis is not required for Hos2 SPG disassembly
To determine whether the recovered proteins were preexisting or newly synthesized, we transferred 1-wk cells into fresh medium in the presence of cycloheximide, an inhibitor of protein synthesis. After 2 h, Hos2 redistributed into the nucleus in nearly 40% of cells, indicating that nuclear Hos2 protein in the recovering cells was not newly synthesized (). Furthermore, even in the presence of cycloheximide, only a small proportion of cells still contained Hos2 SPGs, suggesting that disassembly of these granules was translation-independent. However, unlike the typical nuclear diffusion observed in cells without cycloheximide treatment, Hos2 molecules in many drug-treated cells (56%) remained in the cytosol without a significant change in total protein quantity. It is therefore likely that interference with protein translation leads to inefficient translocation of proteins back into the nucleus.
Glucose is a critical factor determining assembly and disassembly of Hos2 SPGs
A previous study showed that metabolic status controls the entry into and exit from the quiescent state (Laporte et al., 2011
). To understand which nutrients or chemicals trigger assembly of Hos2 SPGs, log-phase cells were resuspended in conditioned medium (from 2-wk cultures), yeast–peptone medium (YEP, rich medium but lacking any carbon source), H2
O, or H2
O plus 2% glucose. Cells were able to form Hos2 SPGs in the conditioned medium, YEP, or H2
O, but not in H2
O plus 2% glucose (). These results suggest that depletion of glucose is sufficient to induce the Hos2 SPG formation. Nonetheless, the kinetics of Hos2 SPG formation in different conditions is not exactly the same, suggesting that some other factors also may be involved. We also tested the effect of glucose on disassembly of Hos2 SPGs. Stationary-phase cells were resuspended in H2
O plus 2% glucose, complete synthetic medium (CSM), and CSM-glucose, and the Hos2 SPGs were monitored. Hos2 SPGs quickly disassembled within 1 h of suspension in H2
O plus 2% glucose or CSM, but did not disassemble in CSM-glucose (). Altogether, our results indicate that glucose is a critical factor for assembly or disassembly of Hos2 SPGs.
FIGURE 5: Glucose is a critical factor for assembly and disassembly of Hos2 SPGs. (A) Depletion of glucose is sufficient to induce the Hos2 SPG formation. Log-phase cells were resuspended in conditioned medium (from 2-wk cultures), YEP, H2O or H2O + 2% glucose, (more ...)
Hos2 SPGs do not colocalize with actin bodies or proteasome storage granules
The actin cytoskeleton is central for intracellular protein localization. F-actin and several actin-binding proteins have been found to assemble into cytoplasmic structures called actin bodies in quiescent cells (Sagot et al., 2006
). Actin localization during stationary phase was examined in cells expressing Hos2-GFP. We found that Hos2 SPGs did not colocalize with actin bodies but that many Hos2 SPGs (46 ± 17%, n = 81) partially overlapped with or were located adjacent to actin bodies (). Similar results were obtained for Hst2 and Yca1, but not for Scl1 (10 ± 6%, n = 342), a component of proteasome storage granules, or Edc3 (9 ± 3%, n = 370), a component of P-bodies.
Proteasomes in yeast are also known to reorganize during the stationary phase into cytoplasmic structures called proteasome storage granules (Laporte et al., 2008
). We found that Hos2 did not colocalize with Scl1, indicating that Hos2 SPGs and proteasome storage granules are distinct structures ().
Hos2 proteins colocalize with stress granules during stationary phase but not under heat-shock conditions
It has been reported that the formation of P-bodies and stress granules is triggered by entry into stationary phase in yeast cells (Teixeira et al., 2005
; Brengues and Parker, 2007
). We therefore tested whether there was any interaction between Hos2 SPGs and these two structures. Our results showed that Edc3, a central component of P-bodies, did not colocalize with the Hos2 SPG in 1-wk cells. However, we also observed that a proportion of Hos2 SPGs were adjacent to the Edc3 signal (38 ± 7%, n = 329), implying an interaction between these two types of granules ( and S6). By contrast, Hos2 SPGs colocalized with the components of stress granules (), Pab1 (86 ± 6%, n = 204), Pbp1 (99 ± 1%, n = 202), and Ygr250c (96 ± 2%, n = 219). The formation of stress granules is greatly reduced in pbp1
mutant cells (Buchan et al., 2008
). However, we found that deletion of PBP1
did not affect the formation of Hos2 SPGs in quiescent cells ().
FIGURE 6: Hos2 SPGs do not colocalize with P-bodies but colocalize with stress granules during stationary phase. (A) Hos2 SPGs do not colocalize with P-bodies (Edc3-GFP), but a proportion of Hos2 SPGs (38 ± 7%, n = 329) are interacting with Edc3 dots (white (more ...)
FIGURE 7: SPG formation in different mutants. (A) Formation of Hos2 SPGs is not affected in pbp1 mutant cells. (B) Yca1 is unable to form granules in stationary-phase hsp42 cells. (C) Formation of proteasome storage granules (Scl1-GFP) is not (more ...)
Many other stresses, including heat shock, are known to induce the formation of stress granules (Buchan and Parker, 2009
). To clarify whether Hos2 also relocalizes into stress granules upon heat shock, we incubated log-phase cells at 37°C or 46°C. No alteration in the localization of Hos2 was observed at 37°C for up to 2 h. On the other hand, robust heat shock at 46°C induced the formation of cytoplasmic punctate foci of Hos2 within 15 min (). The pattern of these punctate foci was different from that observed during quiescence, as they consisted of a larger number of tiny foci scattered in the cytoplasm, rather than one to two large, bright dots. Although heat shock–induced stress granules also display a punctate pattern (Grousl et al., 2009
), there was no apparent colocalization between these two types of foci (). These results suggest that Hos2 SPGs (and starvation stress granules) have distinct structures from previously characterized heat shock–induced stress granules.
Heat-shock proteins Hsp26 and Hsp42 colocalize with Hos2 SPGs in quiescent cells
Molecular chaperones are essential for the assembly and maintenance of many multiprotein complexes (Ellis and Minton, 2006
; Dezwaan and Freeman, 2008
; Gong et al., 2009
). Previous studies have shown that the expression levels of two small heat-shock proteins, Hsp26 and Hsp42, are highly induced in stationary phase (DeRisi et al., 1997
; Gasch et al., 2000
). Thus we decided to examine whether these two chaperones play a role in the formation of Hos2 SPGs. In log-phase cells, both Hsp26 and Hsp42 proteins diffused in the cytosol. On entry into stationary phase, these proteins aggregated into one or a few bright dots. When protein localization was examined in cells expressing both Hos2-mCherry and Hsp26-GFP or Hsp42-GFP, we observed that Hos2 SPGs were highly colocalized with Hsp26 (90 ± 8%, n = 500) and Hsp42 (99 ± 1%, n = 1053; ). This result suggests that both Hsp26 and Hsp42 participate in the formation of Hos2 SPGs.
FIGURE 8: Hsp26 and Hsp42 colocalize with Hos2 SPGs. Cells expressing Hos2-mCherry and Hsp42-GFP or Hsp26-GFP were grown to different stages, and the kinetics of SPG assembly and disassembly were analyzed. (A and B) Both Hsp42-GFP (99 ± 1%, n = 1053) and (more ...)
We subsequently compared the time course of Hos2 SPG formation in cells collected during a 1-wk interval. The proportion of cells exhibiting Hsp26 or Hsp42 dots increased rapidly within the first 3 d of culturing (). In particular, the percentage of cells with Hsp42 dots was nearly identical at 2 d and at 1 wk, indicating that almost all Hsp42 observed in 1-wk cells had already formed within the first 2 d. In contrast to the Hsp26 and Hsp42 signals, which escalated rapidly, Hos2 SPGs formed slowly during the first 2 d of growth; however, formation of Hos2 SPGs significantly increased afterward (). These data reveal that Hsp26 and Hsp42 assemble into SPGs before Hos2 during stationary phase.
Next we examined the behavior of Hsp26 and Hsp42 proteins during cell recovery. After 1-wk cells were provided with nutrients, Hsp26 dots gradually disappeared within 100 min, with a rate similar to that of the disappearance of Hos2 dots (). In contrast, most of the Hsp42 signal was sustained for the duration of the 120-min recovery time (during which more than 50% of cells rebudded; ). Although cells did not rebud until Hos2 had fully relocated, the onset of rebudding did not require the disassembly of the Hsp42 granules. Overall, Hsp26 and Hsp42 granules assembled earlier and disassembled later than Hos2 SPGs, raising the possibility that these two chaperones might serve as scaffolding proteins in the assembly of Hos2 SPGs.
Formation of Hos2 SPGs and stress granules during stationary phase is Hsp42-dependent
The localization pattern of Hsp26 and Hsp42 prompted us to investigate the effect of these two chaperones on the formation of Hos2 SPGs. As shown in , hsp26
cells exhibited no defects in Hos2 SPG formation. In contrast, Hos2 relocalized from the nucleus to the cytoplasm in hsp42
mutant cells, but was unable to assemble into typical SPGs during stationary phase (). The proportion of quiescent cells possessing detectable cytoplasmic Hos2 SPGs was reduced from 88 ± 8% in wild-type cells to 10 ± 2% in hsp42
cells (). Even though some dot-like structures could still be observed in a minority of hsp42
cells, their average size and fluorescence intensity were reduced when compared with wild-type cells (). In hsp42
cells, Yca1 also failed to reorganize into SPGs during stationary phase (). Only 16 ± 7% of quiescent mutant cells had detectable Yca1 SPGs, in contrast to 65 ± 7% of wild-type cells. Again, the dot size and dot intensity of Yca1-GFP were reduced.
FIGURE 9: Formation of Hos2 SPGs depends on Hsp42. (A) Hos2 SPG formation is not affected in stationary-phase hsp26 cells. (B) Hos2 and Pab1 (a component of stress granules) are unable to form granules in stationary-phase hsp42 cells. Scale bars: (more ...)
The above data, together with our prior results showing colocalization of stress granules and Hos2 SPGs in stationary-phase cells, led us to question whether Hsp42 is also critical for the assembly of starvation stress granules. We observed that Pab1 failed to assemble into granules in most hsp42
quiescent cells (). In the few cells that contained visible stress granules, both the dot size and dot intensity were largely decreased (), similar to those seen in Hos2 SPGs.
To examine whether Hsp42 is required for the formation of other types of SPGs, we checked for the formation of proteasome storage granules using Scl1-GFP–expressing cells. As shown in , deletion of HSP42 did not cause any defect in the formation of proteasome storage granules in quiescence. Our results indicate that the small heat-shock protein Hsp42 plays a central and specific role in Hos2 SPG assembly.
Finally, we asked whether deletion of HOS2 would affect the formation of SPGs or P-bodies. In hos2 mutant cells, we did not observe any obvious defect in the formation of Hsp42, Hsp26, Pbp1, Pab1, and Dcp2 dots (Figure S7). Nonetheless, it remains possible that Hos2 has some subtle effects on these structures that cannot be detected by our current assays.