The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers
- University of Zurich, Switzerland
- ETH Zurich, Switzerland
Figures
Figure 1
Nog1DN accumulates on a cytoplasmic pre-60S.
(A) Domain organization and location of Nog1 on a pre-60S (PDB-3JCT). NTD: N-terminal domain; CTT: C-terminal tail. The black arrow points to the G223A mutation in the G3 motif within the G-domain …
Figure 2 with 1 supplement
Association of assembly factors with pre-60S at different maturation stages revealed by SWATH-MS.
Pre-60S particles representing nucleolar (Ssf1-TAP), nuclear (Rix1-TAP), nuclear to cytoplasmic (Arx1-TAP) and cytoplasmic (Lsg1-TAP) stages were analyzed by SWATH-MS. A heat map was generated on …
Figure 2—figure supplement 1
Co-enrichment of assembly factors with a maturing pre-60S subunit.
60S pre-ribosomes representing different maturation stages: nucleolar (Ssf1-TAP), nuclear (Rix1-TAP), nuclear and cytoplasmic (Arx1-TAP) and cytoplasmic (Lsg1-TAP) were purified, and selected …
Figure 3
Nog1DN impairs cytoplasmic maturation of a pre-60S.
(A) Current model for the cytoplasmic maturation pathway of a pre-60S. A cytoplasmic pre-60S at different maturation stages is represented as a cartoon. Energy-consuming assembly factors are …
Figure 4
Nog1DN impairs stalk assembly and terminal maturation steps.
(A) Cryo-EM structure (PDB-3JCT) of a Nog1-containing pre-60S particle, depicting the interaction between Rlp24 and Nog1. (B) A RLP24-TAP strain expressing NOG1 or NOG1DN under the control of GAL1 …
Figure 5
Nog1DN does not impair PET maturation and quality control.
(A) Drg1DN impairs Yvh1 and Rei1 recruitment, whereas Nog1DN impairs only Yvh1 and not Rei1 recruitment. Lsg1-TAP was co-transformed with plasmids each expressing copper-inducible Drg1 or Drg1DN and …
Figure 6
The Nog1-CTT:Rlp24 complex negatively regulates PET maturation.
(A) Domain organization of Nog1 and Rlp24 showing interaction surfaces, and sites for truncation analyses. (B) Growth analysis of Nog1 truncations. Left panel: a Nog1 shuffle strain was transformed …
Figure 7
Nog1DN blocks the terminal cytoplasmic maturation steps.
(A) Nog1DN impairs recycling of Mrt4 and Tif6. The mrt4Δyvh1Δ strain was co-transformed with plasmids encoding Mrt4-GFP or Mrt4G68E-GFP and Yvh1 or galactose-inducible Nog1DN, respectively. To …
Figure 8
A revised model for cytoplasmic maturation of the pre-60S.
Drg1 initiates the 60S cytoplasmic maturation pathway by binding to and releasing Rlp24 from the pre-60S bound Rlp24:Nog1-CTT complex in an ATP-dependent manner (State I to State II). Removal of …
Author response image 1
Functional analyses of yeast mutants expressing Nog1-GFP and Nog1-FLAG.
(A) Polysome profile analysis of Nog1-GFP. Wild-type and Nog1-GFP strains were grown in YPD at 30°C to mid-log-phase. Cell extracts were prepared after cycloheximide treatment and subjected to …
Author response image 2
Arb1 location on a mature 60S clashes with Nmd3 binding site on a pre-60S.
Cryo-EM structures of Nmd3-containing (PDB-6RZZ, Kargas et al., 2019) pre-60S and Arb1-containing (PDB-6R84, Su et al., 2019) mature 60S subunit were superimposed. Surprisingly, Arb1 binds to a …
Additional files
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Supplementary file 1
List of yeast strains used in this study.
- https://cdn.elifesciences.org/articles/52474/elife-52474-supp1-v2.docx
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Supplementary file 2
List of plasmids used in this study.
- https://cdn.elifesciences.org/articles/52474/elife-52474-supp2-v2.docx
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Supplementary file 3
Quantification of microscopy data.
The average fraction of >1000 cells showing stronger nuclear fluorescence than cytoplasmic (fn) from three independent experiments.
- https://cdn.elifesciences.org/articles/52474/elife-52474-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52474/elife-52474-transrepform-v2.pdf
