Fig 1: The N-terminal low complexity and PUF domains of Puf3 are critical for interaction with Pbp1.A. Schematic representation of Puf3 and deletion mutants used. B. Decreased interaction between Puf3 and Pbp1 is observed when the N-terminal LCD or PUF domain is lacking. Cells with endogenously tagged Pbp1 and Puf3 were grown in YPD and then washed and resuspended in YPL. Samples were collected after 3 h in YPL, after which Flag-immunoprecipitation was performed.–denotes negative control lacking Flag-tag. Note: total protein amounts are doubled for Puf3 NtΔ and Puf3 PUFΔ samples as compared to the others, as the expression of these two mutants is reduced (see Input). C. Cox2 protein levels are decreased in Puf3 NtΔ and Puf3 PUFΔ mutants in respiratory conditions. Cells were grown to log phase in YPD and then switched to YPL. Samples were collected after 3 h in YPL, quenched, followed by protein extraction. Equal amounts of protein were assessed by immunoblot for Cox2, Por1, Rpn10, and G6pdh levels. Note that NtΔ and PUFΔ mutants of Puf3 are unstable and expressed at lower levels compared to WT. Transcript levels are shown in S5 Fig.
Fig 2: Increased amounts of Puf3 interact with Pbp1 under respiratory conditions.A. Only proteins whose synthesis is dependent on Puf3-target mRNAs (Cox2) are decreased in puf3Δ, pbp1Δ, and pbp1Δpuf3Δ cells as compared to WT. Cells were grown to log phase in YPD, then washed and resuspended in YPL. Samples were collected before (0 h) and after switch to YPL (3 h), quenched, followed by protein extraction. Equal amounts of protein were assayed by immunoblot for Cox2, Por1, Atp2, Rpn10 and G6pdh levels. How transcript levels respond in the double mutant is shown in S4 Fig. B. Increased amounts of Puf3 are associated with Pbp1 in respiratory conditions. Cells with epitope-tagged Pbp1 and Puf3 were grown in YPD and then washed and resuspended in YPL. Samples were collected at the indicated time points, after which Flag immunoprecipitation was performed.–denotes negative control cells lacking a Flag-tag.
Fig 3: Translational regulation of sulfur metabolism genes via methylation multiplicity(A) Change of translation efficiency (TE) under methionine-replete and methionine-starvation conditions. A 10% false discovery rate (FDR) (−log10(Padj) ≥1) and 2-fold change of TE (log2(TE fold change) ≥1 or log2(TE fold change) ≤ −1) are considered significant, and genes with significantly changed TE are highlighted in black.(B) Representative tracks of ribosome footprint (RFP) and mRNA for JLP1, YCT1, MET3, and RPN10. Two biological replicates for each genotype are shown, and tracks are comparable only within each RFP or RNA group.(C) Simplified schematic of yeast sulfur metabolism. Highlighted are proteins whose transcripts are translated with significantly lower TE in the D87E mutant under methionine-replete conditions.(D) Impact of methionine starvation on TE and mRNA levels of sulfur metabolism genes listed in (C).The p values were calculated using two-sided Mann-Whitney test. ****p < 0.0001.See also Figures S5–S7 and Tables S1, S2, and S3.
Fig 4: Met30 and Met4 response to sulfur starvation and repletion under respiratory growth conditions. a) Schematic of experimental regimen used throughout this study. All sulfur sources were depleted for the indicated times, followed by supplementation of the designated sulfur sources to the same culture for the indicated times. b) Western blot analysis of Met30 and Met4 over the sulfur starvation time course. Yeast containing endogenously tagged Met30 and Met4 were cultured in rich lactate media (Rich) overnight to mid-log phase before switching cells to sulfur-free lactate media (−sulfur) for 1 h, followed by the addition of a mix of the sulfur-containing metabolites methionine, homocysteine, and cysteine at 0.5 mM each (+Met/Cys/Hcy). Rpn10 is used as the loading control. The blot shown is representative of 3 replicate experiments. c) Expression of MET gene transcript levels was assessed by qPCR over the time course shown in (a). Data are presented as mean and SEM of technical triplicates. d) Levels of key sulfur metabolites were measured over the same time course as in (a) and (b), as determined by LC-MS/MS. Data represent the mean and SD of 2 biological replicates.
Fig 5: C-terminal, low complexity regions of Pbp1 mediate its interaction with Puf3.A. Schematic representation of Pbp1 and the deletion mutants used. B. Interaction between Puf3 and Pbp1 lacking its mid-section (midΔ) or C-terminal LCD (LCDΔ) is decreased in respiratory conditions. Cells with epitope-tagged Pbp1 and Puf3 were grown in YPD and then washed and resuspended in YPL. Samples were collected at the indicated time points, after which Flag immunoprecipitation was performed.–denotes negative control lacking a Flag-tag, but including HA-tag. * denotes non-specific band. C. Cox2 protein levels are decreased in Pbp1 midΔ, LCDΔ, and pbp1Δ in respiratory conditions. Cells were grown to log phase in YPD, then washed and resuspended in YPL. Samples were collected after 3 h in YPL, quenched, followed by protein extraction. Equal amounts of protein were assessed by immunoblot for Cox2, Por1, Atp2, Pbp1, Rpn10, and G6pdh levels. Transcript levels are shown in S5 Fig. D. Growth curves of the indicated strains in YPD or YPL media. E. Schematic representation of Pbp1 LCD-mutants used. Methionine residues within this key region of the LCD (M591, M595, M605, M606, M614, M616, M618, M625) were mutated to serine (M8S), phenylalanine (M8F), or tyrosine (M8Y). Pbp1 M8S forms weaker assemblies than WT, whereas M8F and M8Y both form stronger assemblies. F. Pbp1-Puf3 interaction is decreased in the M8S mutant. Cells expressing the indicated, epitope-tagged variants of Pbp1 and Puf3 were grown in YPD and then washed and resuspended in YPL. Samples were collected at 3 h in YPL, after which Flag-immunoprecipitation was performed.–denotes negative control lacking Flag-tag. G. Cox2 protein levels are decreased in M8S, LCD, and pbp1Δ mutants. Cells expressing the indicated epitope-tagged variants of Pbp1 were grown to log phase in YPD, then washed and resuspended in YPL. After 3 h, cells were harvested and quenched, followed by protein extraction. Equal amounts of protein were assessed by immunoblot for Cox2, Por1, Pbp1, and G6pdh levels.
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