1. Volume 48, Issue 2, Pages 207-218 (October 2012)
Architecture of the Nuclease Module of the Yeast Ccr4-Not Complex: the Not1-Caf1- Ccr4 Interaction 
Jérôme Basquin, Vladimir V. Roudko, Michaela Rode, Claire Basquin, Bertrand Séraphin, Elena Conti 
Molecular Cell 
Volume 48, Issue 2, Pages (October 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 The Caf1-Ccr4-Binding Domain of Not1 Is Essential In Vivo
(A) Schematic representation of the domain arrangement of yeast Not1, Caf1, and Ccr4. The Not1154–753 and Not1754–1000 domains were identified biochemically (Figure S1) and are shown in red and orange, respectively. Other predicted structured regions in Not1 are colored in gray. The nuclease domain of Caf1 is in blue, and the LRR and nuclease domains of Ccr4 in pink. The N-terminal region of yeast Ccr4 present in the crystal structure reported in this manuscript is shaded in pink. Unstructured regions are in white.
(B) Analysis of the ability of Not1 truncation mutants to complement a Not1 deletion. TRP1-marked plasmids encoding different truncations of Not1 (fused to the TAP tag) are schematized on the left. The plasmids were introduced in strain T26N28 carrying a chromosomal deletion of NOT1 complemented by a wild-type copy of the gene on a URA3-marked plasmid. Complementation was assayed by monitoring growth on FOA, growth on –Trp media serving as a control.
(C) Analysis of the levels of Not1 protein accumulating in the wild-type strain and mutants expressing truncated forms of the protein. Total proteins extracted from the various strains were fractionated by SDS-PAGE, transferred to a membrane, and detected through the TAP tag present at the C terminus of the proteins.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 2 Structure of the Not1754–1000-Caf1-Ccr4 Complex
(A) The complex is shown in two different orientations related by a 180° rotation around a vertical axis. Not1 is in orange, Caf1 in blue, and Ccr4 in pink. On the right, the complex is shown superposed to the structure of the nuclease domain of the human Ccr4 ortholog CNOT6L (in gray, PDB 3NGQ). The five LRR motifs and the N and C termini are labeled. Disordered loops are indicated with a dotted line. Residues at the active sites of Caf1 and Ccr4 are shown in stick representation. All structure figures in this manuscript were generated with Pymol (
(B) Surface representation of the complex in similar orientations as in (A) and including the superposed CNOT6L nuclease domain. The surface is colored according to sequence conservation, ranging from white (variable residues) to dark orange (invariant residues). The conservation was mapped with the program Consurf (Ashkenazy et al., 2010) based on the alignment in Figure S2.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 3 The Interactions in Not1754–1000-Caf1-Ccr4 Are Conserved
The figure shows four enlarged regions of the complex discussed in the text.
(A) On the left, the crystal structure of the Not1754–1000 domain is shown in orange superposed to a MIF4G domain of UPF2 (Kadlec et al., 2004, in gray). The five HEAT repeats are labeled and the N- and C-terminal segments are indicated. On the right, Not1754–1000 is shown in the same orientation as a surface representation colored according to sequence conservation, ranging from white (variable) to dark orange (conserved), as described for Figure 2B.
(B) Zoom-in showing a subset of residues in stick representation at the Not1-Caf1 interface. Residues mutated in the functional assays in Figure 4 are indicated.
(C) Zoom-in showing a subset of residues in stick representation at the Caf1-Ccr4 interface. Residues mutated in the functional assays in Figure 4 are labeled. Also shown are residues reported in previous mutagenesis studies (Asp357, Phe358) (Clark et al., 2004) and residues at the Caf1 active site (Glu190, etc.).
(D) Zoom-in of the LRR-nuclease interaction of Ccr4. Hydrophobic residues at the interface are indicated, as well as conserved residues exposed on the surface.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 4
The Interactions in Not1754–1000-Caf1-Ccr4 Are Essential for Normal Growth and mRNA Decay
(A–C) Coimmunoprecipitation was used to assay Not1-Caf1-Ccr4 interactions.
(A) VSV-tagged wild-type or mutant Caf1 present in strains expressing wild-type HA-tagged Not1 and HA-tagged Ccr4 were immunoprecipitated with anti-VSV antibodies. Parallel anti-VSV immunoprecipitations were performed from strains expressing VSV-tagged wild-type Caf1, HA-tagged mutant Not1, and wild-type HA-tagged Ccr4. Proteins present in the pellet fraction were detected by western blotting with anti-HA (Ccr4 and Not1) and anti-VSV (Caf1) antibodies.
(B) TAP-tagged wild-type Ccr4 (left lanes) or TAP-tagged Not1 (right lanes) present in strains expressing either wild-type or mutant VSV-tagged Caf1 were immunoprecipitated on IgG beads. Proteins present in the pellet fraction were detected by western blotting with anti-VSV antibodies (Caf1) and peroxidase-anti-peroxidase complex (Ccr4 or Not1).
(C) TAP-tagged wild-type Not1 present in strains expressing either VSV-tagged Ccr4 and/or VSV-tagged Caf1 was immunoprecipitated. Either wild-type or the indicated mutant forms of Ccr4 were used for this experiment. Proteins present in the pellet fraction were detected by western blotting with anti-VSV antibodies (Ccr4 and Caf1) and peroxidase-anti-peroxidase complex (Not1).
(D) Growth phenotypes resulting from mutations of interface residues. Plasmids encoding the Caf1, Ccr4, and Not1 mutants were introduced in strains deleted of the cognate wild-type gene, either directly (Caf1 and Ccr4) or by plasmid shuffling (Not1). Growth of the resulting strain was assayed on YPDA media at 30°C and 37°C.
(E) The plasmid encoding the MFA2pG reporter was introduced in the wild-type yeast strain, isogenic deletion strains lacking Ccr4 or Caf1 and point mutant of interface residues. The decay of the MFA2 mRNA was monitored in chase experiments. The MFA2 mRNA, decay intermediates and U5 small nuclear RNA were detected by northern blotting after fractionation in denaturing polyacrylamide gels.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5 Not1154–753 Is a HEAT-Rrepeat Platform
(A) Crystal structure of Not1154–753 shown in a ribbon representation in red. The HEAT repeats and the ordered N and C termini are labeled. Disordered loops are indicated with a dotted line.
(B) Not1154–753 is shown in the same view as a surface representation colored according to sequence conservation as in Figure 2B.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 6 Structural Organization of the Yeast Ccr4-Not Complex
The scheme recapitulates the information from the crystal structures of Not1754–1000-Caf1-Ccr4 and Not1153–753 reported in this manuscript, combined with structural information from an EM study of the Ccr4-Not complex (Nasertorabi et al., 2011) (Figure S6) and previous interaction studies (reviewed in Collart and Timmers, 2004). Not1 is shown with the two structural arms colored from red (N terminus, residue 1) to yellow (C terminus, residue 2,108). Caf1 and Ccr4 are shown in blue and pink, with stars indicating their active sites. The Not2, Not3, Not4, and Not5 proteins that are part of the C-terminal “Not module” are shown in gray. Not shown here are Caf40 and Caf130, for which the interacting regions within the complex have yet to be precisely identified. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions