Analysis of stress granule assembly in Schizosaccharomyces pombe

Abstract

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.

FIGURE 1.

G3BP-like protein Nxt3 as a component of fission yeast SGs. (A) Schematic representation of the domain structures of S. pombe Nxt3, S. cerevisiae Bre5, and human G3BP1. (NTF2) Nuclear transport factor 2–like domain; (RRM) RNA recognition motif. (B) Nxt3-GFP was visualized after growth to mid-logarithmic phase at 30°C (no treatment), or after deprivation of glucose or nitrogen for 1 and 3 h, respectively, or after exposure to 1 M KCl and after a shift to 42°C for 15 min. One hundred micrograms per milliliter (100 μg/mL) CHX was added 1 min before the shift to 42°C. (C) Merged images of fluorescence micrographs showing Pabp-mCherry (red) and Nxt3-GFP (green) localization in living cells grown at 30°C, and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (Bar) 5 μm.

FIGURE 2.

Ubp3 as an interaction protein of Nxt3. (A) Schematic representation of the domain structures of S. pombe Ubp3, S. cerevisiae Ubp3, and human Usp10. (B) Tandem affinity purification (TAP)–tag pull-down of Nxt3 proteins resolved by SDS-PAGE were visualized by silver staining. (Right) Identity of constituent proteins (identified by MALDI MS/MS analysis of individual bands). (*) Degradation products of Nxt3. (C) TAP-tag pull-down of Ubp3 proteins from strain expressing Nxt3-HA fusion proteins incubated at 42°C for the time indicated were separated by SDS-PAGE and subjected to Western blotting using anti-PAP and anti-HA antibodies. (D) Merged images of fluorescence micrographs showing Ubp3-tdTomato (red) and Nxt3-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C. (Bar) 5 μm.

FIGURE 3.

Assembly of Nxt3 and Ubp3 into SGs does not require their forming a complex with each other. (A) The Ubp3-GFP protein level was decreased in the nxt3 mutant, and vice versa. Whole-cell protein extracts from the indicated strains expressing Nxt3 or Ubp3 GFP-fusion proteins were separated by SDS-PAGE and subjected to Western blotting using anti-GFP and anti-tubulin (loading control) antibodies. Relative intensities of the indicated proteins were indicated beneath each lane. (B) Fluorescence micrographs of the indicated mutant strains expressing Nxt3 or Ubp3 GFP-fusion proteins grown at 30°C and after a 15-min incubation at 42°C. (Bar) 5 μm.

FIGURE 4.

Nxt3 and Ubp3 are dispensable for the formation of fission yeast SGs. (A) Fluorescence micrographs of the indicated strains expressing Pabp-GFP protein grown at 30°C and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (Bar) 5 μm. (B) Pabp-GFP was visualized in strains grown at 30°C (time 0) or after a shift to 42°C for the time indicated. (C) Localization of Pabp-GFP after a 15-min incubation at 42°C (time 0) followed by a shift back to 30°C for 30 min. (D) Localization of the Nxt3 and Ubp3 GFP-fusion proteins under the control of its own promoter (Pnxt3 and Pubp3) or overexpressed by the nmt1 promoter (Pnmt1). (E) Whole-cell protein extracts from cells expressing Nxt3 or Ubp3 GFP-fusion proteins under the control of its own promoter (Pnxt3 and Pubp3) or overexpressed by the nmt1 promoter (Pnmt1) were separated by SDS-PAGE and subjected to immunoblotting using anti-GFP antibodies to reveal the GFP-fusion proteins. Antibodies against α-tubulin were used as controls.

FIGURE 5.

TIA-like proteins Csx1 and Cxr1 are not required for the assembly of fission yeast SGs. (A) Schematic representation of the domain structures of TIA-like proteins S. pombe Csx1 and Cxr1, S. cerevisiae Pub1 and Ngr1, and human TIA-1 and TIA-R. (RRM) RNA recognition motif. (B) Cladogram showing the relationship between S. pombe Cxr1 and TIA-like proteins from S. cerevisiae to human. The length of each pair of branches represents the distance between sequence pairs. (C) Merged images of fluorescence micrographs showing Pabp-mCherry (red) and TIA-like proteins' (Csx1 and Cxr1; green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (Bar) 5 μm. (D) Fluorescence micrographs of the indicated mutant strains expressing Pabp-GFP protein grown at 30°C and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (E) Localization of the Csx1 GFP-fusion protein under the control of its own promoter (Pcsx1) or overexpressed by the nmt1 promoter (Pnmt1). (F) Localization of the Cxr1 GFP-fusion protein under the control of its own promoter (Pcxr1) or overexpressed by the nmt1 promoter (Pnmt1).

FIGURE 6.

The ATXN2 homolog is not required for the assembly of fission yeast SGs. (A) Schematic representation of the domain structures of S. pombe Ath1, S. cerevisiae Pbp1, and human ATXN2. (B) Merged images of fluorescence micrographs showing Pabp-mCherry (red) and Ath1-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (Bar) 5 μm. (C) Fluorescence micrographs of the indicated mutant strains expressing Pabp-GFP protein grown at 30°C and after a 15-min incubation at 42°C, or deprivation of glucose for 1 h. (D) Localization of the Ath1 GFP-fusion protein under the control of its own promoter (Path1) or overexpressed by the nmt1 promoter (Pnmt1).

FIGURE 7.

Ubp3-Nxt3 associates with polyribosome. (A) Cytosolic native extracts from CHX-treated yeast cells grown at 30°C were separated on 7%–47% sucrose gradients. The distribution of the rRNA is shown by absorption profiles at 260 nm and the distribution of proteins of interest by Western blotting after denaturing gel electrophoresis. (B) Cytosolic native extracts from CHX-treated yeast strains as indicated were separated on 7%–47% sucrose gradients. The distribution of the rRNA is shown by absorption profiles at 260 nm.