A quantitative inventory of yeast P body proteins reveals principles of composition and specificity
- Department of Biophysics, Howard Hughes Medical Institute, UT Southwestern Medical Center, United States
- Department of Biochemistry, Howard Hughes Medical Institute, University of Colorado, United States
Figures
Figure 1 with 3 supplements
Seven proteins are highly concentrated in P bodies.
(A) Partition coefficients (PCs) of 19 P body proteins. Plots show PCs and mean values (blue lines)± standard error of the mean (SEM). (B) Absolute concentrations in P bodies of 19 P body proteins. …
Figure 1—figure supplement 1
Punctate localization of GFP tagged proteins and co-localization with P body marker.
(A) Confocal fluorescence microscopy images of 31 P body proteins fused to GFP expressed in dcp1Δ strain in log-phase. Scale bar, 5 µm. (B) Fluorescence microscopy images of yeast co-expressing …
Figure 1—figure supplement 2
Quantifications of protein concentrations using fluorescence intensities.
(A) Measure maximum intensity of P body and average intensity of cytoplasm. (B) Measure size of P body by drawing a line across the P body and measure the full width at half maximum intensity …
Figure 1—figure supplement 3
Verification of quantitative measurements.
(A) A C-terminal GFP tag does not affect protein partitioning into P bodies. Plots show PCs (black dots) and mean values (red lines)± SEM when Edc3 and Dhh1 tagged at their C-termini with either GFP …
Figure 2 with 4 supplements
Highly concentrated proteins tend to have slow dynamics.
Exchange rate (A) or fractional recovery (B) was plotted as a function of absolute concentrations in P bodies, respectively (mean ± SEM). HC and LC proteins are indicated by red and black symbols, …
Figure 2—figure supplement 1
FRAP recovery curves of 19 P body proteins.
FRAP recovery curves of 19 P body proteins reported as average of 16–25 P bodies ± SD.
Figure 2—figure supplement 2
Slow dynamics are not caused by smaller fluorescence pool in cytoplasm.
(A/B) Exchange rate (A) or fractional recovery (B) is not correlated with protein concentrations in cytoplasm. Exchange rate or fractional recovery (mean ± SEM) were plotted as a function of …
Figure 2—figure supplement 3
Partitioning and dynamics of P body proteins in wild type strains under glucose starvation are qualitatively similar to dcp1Δ strains.
(A/B) Partition coefficients (A) or absolute concentrations (B) in P bodies in wild type strains under 30–60 min glucose starvation. Plots show PC values, and absolute concentrations in P bodies …
Figure 2—figure supplement 4
Protein-protein and protein-RNA connections among regular P body proteins.
Protein-protein and protein-RNA interactions are summarized from literatures as shown in Supplementary file 3. Red, HC P body proteins. Black, LC P body proteins. Grey, 12 P body proteins that …
Figure 3
Partitioning of proteins into P bodies is correlated.
(A) Schematics of calculations of ratios of paired and randomized proteins. Paired ones are ratios of X-GFP to Dcp2-mCherry in the same P bodies where i = i’. Randomized pairing are achieved by …
Figure 4 with 1 supplement
P bodies do not strongly sequester their resident proteins.
Plots show fractions in visible P bodies (black dots) and mean values (red lines)± SEM in mid-log phase without any cellular stress. Each dot represents fraction of protein in visible P bodies …
Figure 4—figure supplement 1
P bodies do not strongly sequester their resident proteins.
(A) Fractions of total protein localized to P bodies (black dots) and mean values (red lines)± SEM in dcp1Δ strains. Cells were grown to mid-log phase followed by 4 hr glucose starvation. (B) …
Figure 5 with 1 supplement
Elements controlling Dcp2 partitioning and dynamics are distributed across the protein, and contribute to specific recruitment to biomolecular condensates.
(A) Schematics of domain architecture of Dcp2 FL and mutants. Red, N-terminal domain (NTD). Orange, HLM1. Blue, other 10 HLMs in C-terminal domain. Grey, inactivated HLMs. (B/C) Partition …
Figure 5—figure supplement 1
Partitioning of Dcp2 variants.
(A) Representative images of dcp1Δdcp2Δ yeast expressing Edc3-mCherry as a P body marker. Scale bar, 5 µm. Plot shows size of P body in dcp1Δdcp2Δ (n = 34) and dcp1Δ strains (n = 40)± SEM. (B) …
Figure 6 with 1 supplement
Partitioning of Dcp2 into P bodies can be saturated.
(A) Schematics of domain architecture of Dcp2 FL, Dcp2 FL-H1, Dcp2C Δ5H, and Dcp2C Δ5H-H1. (B) Representative images showing dcp1Δdcp2Δ yeast strains expressing GFP tagged Dcp2 FL, Dcp2 FL-H1, Dcp2C …
Figure 6—figure supplement 1
Average cellular concentrations of Dcp2 FL, Dcp2 FL-H1, Dcp2C Δ5H, and Dcp2C Δ5H-H1 in dcp1Δdcp2Δ strain expressing Edc3-mCherry, and mean values (red lines)± SD.
Figure 7 with 1 supplement
RNA binding and turnover affect Dcp2 partitioning and dynamics.
(A) Schematics of domain architecture of Dcp2 300, Dcp2 300 AAAA, Dcp2ΔH1 and Dcp2ΔH1 WD. (B) Representative images of dcp1Δdcp2Δ yeast strain expressing GFP tagged Dcp2 300 and Dcp2 300 AAAA. Scale …
Figure 7—figure supplement 1
Mutant Dcp2 proteins express at similar levels.
(A) Western blot of Dcp2 300 and Dcp2 300 AAAA in dcp1Δdcp2Δ strain shows that they expressed at similar levels. (B) Western blot of Dcp2ΔH1 and Dcp2ΔH1 WD in dcp2Δ strain shows that they expressed …
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/56525/elife-56525-supp1-v2.xlsx
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Supplementary file 2
Dynamics and partitioning of P body proteins.
- https://cdn.elifesciences.org/articles/56525/elife-56525-supp2-v2.docx
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Supplementary file 3
Protein-Protein and Protein-RNA interactions among P body proteins.
- https://cdn.elifesciences.org/articles/56525/elife-56525-supp3-v2.xlsx
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Supplementary file 4
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/56525/elife-56525-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56525/elife-56525-transrepform-v2.docx
