Localization to, and effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on stress granules in Saccharomyces cerevisiae

Abstract

The regulation of translation and mRNA degradation in eukaryotic cells involves the formation of cytoplasmic mRNP granules referred to as P-bodies and stress granules. The yeast Pbp1 protein and its mammalian ortholog, Ataxin-2, localize to stress granules and promote their formation. In Saccharomyces cerevisiae, Pbp1 also interacts with the Pab1, Lsm12, Pbp4, and Dhh1 proteins. In this work, we determined whether these Pbp1 interacting proteins also accumulated in stress granules and/or could affect their formation. These experiments revealed the following observations. First, the Lsm12, Pbp4, and Dhh1 proteins all accumulate in stress granules, whereas only the Dhh1 protein is a constitutive P-body component. Second, deletion or over-expression of the Pbp4 and Lsm12 proteins did not dramatically affect the formation of stress granules or P-bodies. In contrast, Pbp1 and Dhh1 over-expression inhibits cell growth, and for Dhh1, leads to the accumulation of stress granules. Finally, a strain lacking the Pab1 protein was reduced at forming stress granules, although they could still be detected. This indicates that Pab1 affects, but is not absolutely required for, stress granule formation. These observations offer new insight into the function of stress granule components with roles in stress granule assembly and mRNP regulation.

Figure 1. Lsm12 and Pbp4 localize to stress granules.

GFP-tagged Lsm12 and Pbp4 were assessed for their ability to form granules that co-localize with either Edc3-mCherry, a P-body marker, or with Pub1-mCherry, a stress granule marker, following ten minutes of glucose deprivation stress.

Figure 2. Dhh1 localizes to both P-bodies and stress granules following glucose deprivation stress.

(A) A strain harboring a Dhh1-GFP integration was transformed with a plasmid containing Edc3-mCherry and Pab1-CFP and assayed for GFP co-localization with mCherry and/or CFP upon glucose deprivation. (B) A closer view of three sample cells, labeled accordingly in A, is shown. Dhh1 co-localized with Edc3-mCherry (gold arrows), Pab1-CFP (purple arrows), and both Edc3-mCherry and Pab1-CFP (white arrows). Dhh1 was also found in independent foci (green arrows).

Figure 3. Lsm12 and Pbp4 are not required for stress granule formation.

lsm12Δ, pbp4Δ, and wild-type strains were transformed with a plasmid containing Edc3-mCherry (P-body marker) and Pab1-GFP (stress granule marker) and assessed for the ability to form either granule following ten minutes of glucose deprivation stress. (SGs denotes stress granules; PBs denotes P-bodies.)

Figure 4. Pab1 promotes, but is not absolutely required for stress granule formation.

pab1Δ spb2Δ, spb2Δ, and wild-type strains (A), as well as pab1Δ pat1-2, pat1-2, and wild-type strains (B), were transformed with a plasmid containing Pbp1-GFP (stress granule marker) and Edc3-mCherry (P-body marker) and analyzed for granule formation following ten minutes of glucose deprivation stress.

Figure 5. Dhh1, Pbp1, and Pab1 over-expression inhibit growth in wild-type and deletion strains.

Proteins were expressed using galactose-inducible promoters on high copy plasmids and assessed for growth in both wild-type and deletion strains on media containing different concentrations of galactose as their carbon source. All plates were incubated at 30°C for five days.

Figure 6. Dhh1 and Pbp1 over-expression trigger Pab1 granule formation in the absence of stress.

Proteins were expressed under galactose-inducible promoters on high copy plasmids in wild-type cells. (A) Cells were assayed for their ability to form P-bodies (Edc3-mCherry) or stress granules (Pab1-GFP) in media containing 2% galactose with 0.25% sucrose. (B) Granules triggered by Dhh1 over-expression are dependent upon actively translating mRNAs. Addition of 100 µg/ml cycloheximide for 30 minutes to block translation following two hours of galactose induction substantially reduced Pab1 foci formation in cells over-expressing Dhh1, while only slightly reducing Pab1 foci in cells over-expressing Pbp1. (C) Pab1 over-expression does not cause granule formation as assessed by the P-body marker, Edc3, and the stress granule marker, Pbp1, in media containing 2% galactose with 0.25% sucrose. (*) denotes underestimate of Pab1 foci due to large string-like aggregation.

Figure 7. Pbp1 is required for proper expression of both Lsm12 and Pbp4.

Likewise, Lsm12 is required for proper expression and granule formation of Pbp4. (A) Deletion - GFP crosses and wild-type GFP strains were assayed for foci formation following ten minutes of glucose deprivation stress. (B) Western analysis of the GFP-tagged proteins was done on whole-cell extracts using an anti-GFP antibody to detect protein levels. It is important to note that the blots have been cropped and the deletion and controls aligned with each other because, while the deletion and control strains were run on the same gel, they were not run in adjacent lanes.