1. Volume 67, Issue 6, Pages 990-1000.e3 (September 2017)
The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation 
Homa Ghalei, Juliette Trepreau, Jason C. Collins, Hari Bhaskaran, Bethany S. Strunk, Katrin Karbstein 
Molecular Cell 
Volume 67, Issue 6, Pages e3 (September 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 990-1000.e3DOI: (10.1016/j.molcel.2017.08.007)
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3. Figure 1 Sordarin Rescues the Phenotypes from Fap7 Depletion
(A) Sordarin stabilizes the rotated state.
(B) Doubling times of Gal::Fap7 cells in yeast extract peptone dextrose (YPD) medium in the presence (right) or absence (left) of 8 μg/mL sordarin. Data are the average of three biological replicates, and error bars indicate SEM. Unpaired t test was used for statistical analysis; ∗∗∗∗p < , ∗∗∗p =
(C) Northern blot analyses from total cellular RNA from Gal::Fap7 cells grown in YPD with or without sordarin for up to 16.5 hr. Pre-18S rRNA (20S) and mature 18S and 25S rRNAs are detected.
(D and E) 10%–50% sucrose gradients from cell lysates of Gal::Fap7 cells grown in glucose for 16 hr in the absence (D) or presence (E) of sordarin. Shown below the absorbance profile at 254 nm are western blots of assembly factors and northern blots of 20S, 18S, and 25S rRNAs. 63% and 50% of 20S rRNA are in the 80S fraction without and with sordarin, respectively.
See also Figure S1.
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4. Figure 2
The Rpl3_H256A Mutation Rescues the Phenotypes from Fap7 Depletion
(A) The Rpl3_H256A mutation stabilizes the rotated state (Meskauskas and Dinman, 2007).
(B) Growth of cells with wild-type Rpl3 or Rpl3-H256A in the presence or absence of Fap7 was compared by 10-fold serial dilutions on YPD or yeast extract peptone galactose (YPgal) plates.
(C) Doubling times of wild-type or Rpl3-H256A cells in the presence or absence of Fap7 in minimal medium containing glucose. The data are the average of three biological replicates, and error bars indicate SEM. Dunnett’s multiple comparisons test was used for statistical analysis; ∗∗∗∗p <
(D) Northern blots of total RNA from cells with wild-type Rpl3 or the Rpl3_H256A mutant grown in the presence or absence of Fap7 and probed for 20S, 18S, and 25S rRNAs. Numbers indicate the relative amount of 20S pre-rRNA to 25S rRNA.
(E and F) Sucrose gradients of whole-cell extracts from Fap7-depleted cells with wild-type Rpl3 (E) or Rpl3_H256A (F). Shown below the absorbance profile at 254 nm are western blots of assembly factors and northern blots of 20S, 18S, and 25S rRNAs. 41% or 21% of 20S rRNA are in the 80S fraction for the WT and H256A, respectively.
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5. Figure 3
The Rpl3_W255C Mutation Confers a Phenotype Similar to that of Fap7 Depletion
(A) The W255C mutation stabilizes the classic state (Meskauskas and Dinman, 2007).
(B) Growth analysis of cells containing wild-type or W255C Rpl3.
(C) Northern blots of pre-18S rRNA (20S) and mature rRNAs (25S and 18S) demonstrate a block in 40S maturation in the Rpl3_W255C mutant.
(D and E) Sucrose gradient and western and northern blot analyses of the fractions from whole-cell extracts of parent (D) and Rpl3_W255C strains (E). 8% or 12% of 20S rRNA are in the 80S fractions for the WT and W255C, respectively.
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6. Figure 4 Fap7 Induces the Rotated State in 80S-like Ribosomes
(A) 80S-like ribosomes purified from Gal::Fap7 cells via Tsr1-TAP were mixed with recombinant proteins and nucleotides as indicated and treated with DMS (plus) or solvent (minus). DMS modification was detected by reverse transcription. Classic and rotated mature 80S complexes were used as controls.
(B and C) Quantitation of the data shown in (A). The data are the average of 2 biological replicates (one for G19S) with 3 technical replicates each, and error bars indicate SEM. Significance for each column was analyzed relative to the 80S-like column with Dunnett’s multiple comparisons test. n.s., non-significant; ∗∗∗∗p < , ∗∗p = , ∗p = (B); ∗∗∗∗p < , ∗∗p = , ∗p = 0.01 (C).
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7. Figure 5 Fap7 Is Required for Release of Dim1 from 80S-like Ribosomes
(A) Fap7 binds Dim1. Shown are Coomassie-stained SDS-PAGE of protein binding assays on amylose beads. IN, input; W, final wash; E, elution. Note that the maltose binding protein (MBP) tag on Dim1 appears to weaken the Fap7⋅Dim1 interaction.
(B) Co-sedimentation assay to study the release of Dim1. Shown are western blots of Dim1, Tsr1-TAP, Pno1, Rpl3, and Rps8 in the bound (pellet [P]) and released (supernatant [S]) fractions of purified 80S-like ribosomes after no treatment or the indicated treatments.
See also Figure S2.
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8. Figure 6
Fap7-Independent Release of Dim1 Leads to Translational Errors
(A) Comparison of doubling times of wild-type (WT, black column) Dim1 or Dim1_EKR (EKR, hatched column) in the presence of WT or mutant Fap7 (G19S and K20R) in minimal medium containing glucose. The data are the average of three biological replicates, and error bars show SEM. Each column was analyzed relative to the WT/WT column with Dunnett’s multiple comparisons test; ∗∗∗∗ = p < The white columns show the expected doubling times when there was no rescue of the G19S/K20R mutations by Dim1_EKR. The height of these columns was calculated by multiplying the observed fold differences for each single mutation (G19S or K20R with EKR).
(B) Northern blots of total RNA from cells with WT or mutant Dim1 (EKR) proteins grown in the presence of WT Fap7 or mutant Fap7 (G19S) and probed for 20S, 18S, and 25S rRNAs. Numbers indicate the relative amount of 20S pre-rRNA to 25S rRNA.
(C and D) Sucrose gradients of whole-cell extracts from Fap7-K20R cells with either wild-type Dim1 (C) or Dim1_EKR (D). Shown below the absorbance profile at 254 nm are western blots of assembly factors and northern blots of 20S, 18S, and 25S rRNAs.
(E) The effects from the Dim1_EKR mutation on the fidelity of start codon recognition, decoding, stop codon recognition, and frameshifting (−1 and +1) were assayed using dual luciferase reporters (Figure S5) in a Gal::Dim1 strain supplemented with plasmids encoding wild-type or mutant Dim1. Shown are the relative error rates of the Dim1_EKR samples relative to Dim1_wild-type samples. The data are the average of 3–6 biological replicates, and error bars indicate the SEM. ∗∗∗p < and ∗∗∗∗p < by unpaired t test.
See also Figures S3–S6.
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9. Figure 7
Model for Fap7-Dependent Probing of the Ability to Adopt the Hybrid State during 40S Maturation
(A) Model for the role of Fap7 and eEF2 in 40S ribosome assembly.
(B) Interplay between Fap7-mediated Dim1 release and quality control. The assembly cascade produces pre-40S ribosomes that are correctly assembled (light gray) and those defective for subunit rotation (dark gray). The defective 40S retain Dim1 because they are deficient in the Fap7-dependent release of Dim1 and will be degraded.
(C) Dim1_EKR allows for bypass of the Fap7-mediated step because Dim1 can be released Fap7-independently, thereby allowing the defective ribosomes to evade degradation and continue maturation.
See also Figure S7.
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