1. Volume 3, Issue 6, Pages 2155-2167 (June 2013)
A Systematic Approach for the Genetic Dissection of Protein Complexes in Living Cells 
Guillaume Diss, Alexandre K. Dubé, Joël Boutin, Isabelle Gagnon-Arsenault, Christian R. Landry 
Cell Reports 
Volume 3, Issue 6, Pages (June 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 2155-2167DOI: (10.1016/j.celrep.2013.05.004)
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3. Figure 1 Genetic Dissection of Protein Interactomes
(A) Changes in PPIs could arise through different, nonexclusive mechanisms. The deletion of gene A (dashed line) could lead to the disruption of the B-C interaction if (i) protein A is an adaptor protein for protein B and C or (ii) protein A stabilizes protein B and/or C (left). The deletion of A could lead to new B-C interactions if (iii) proteins C and A normally compete for B or (iv) a complex can adopt a new configuration upon the deletion of A (right). Empty shapes are absent proteins.
(B) Current model of the architecture of the retromer and the associated transmembrane sorting receptor Vps10p (Seaman et al., 1998).
(C) Current model of the architecture of the NPC. Subcomplexes are organized into five different rings: membrane (red), inner (cyan), linker (yellow), outer (dark blue), and FG-nucleoporins (green) (Alber et al., 2007).
(D) Systematic strain construction. SGA markers are introduced in the yeast deletion collection (4,293 strains). SGA deletion strains are crossed with PCA strains to introduce gene deletions and the DHFR-fused genes into the same haploid background. The resulting haploid strains are crossed to measure PPIs in a deletion background. PPIs can also be measured in strains that are heterozygous for one or two deletions (not shown). Control strains (wild-type, HO deletion) follow the same procedure.
See also Figure S1 and Tables S1, S5, S6, and S7.
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4. Figure 2 Dissection of the Retromer Complex
(A) I scores in the wild-type and mutant genetic backgrounds. Each data point is the average of 12 independent measurements of strains constructed and tested independently. In the top and right margins are the bimodal distributions of the I scores. P scores measure the difference between the mutant and wild-type I scores. Significant hits (p < 0.006, FDR = 1%) are colored according to their absolute P score value.
(B) Confirmation of gene deletion effects on PPIs by spot dilution assays. Methotrexate (MTX) is the inhibitor of the endogenous yeast DHFR, and DMSO is the MTX solvent.
(C) Result confirmation by coimmunoprecipitation (top) and the corresponding I score measured by PCA (bottom). Bars represent 95% confidence intervals. The dotted line represents the 95% positive PPI threshold.
WT, wild-type.
See also Figure S2 and Table S2.
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5. Figure 3 Genetic Dissection of the Retromer Interactome
(A) Distribution of P scores in homozygous and heterozygous backgrounds (single- and double-heterozygous genotypes).
(B) Distribution of P scores in heterozygous backgrounds with zero, one, or two allelic competitors (AC).
(C) The removal of allelic competitors of the DHFR-fragment tagged proteins (represented with an asterisk) leads to an increase in the proportion of reconstituted DHFR and thus to an increase in growth rate. Strain genotypes are shown on the left and the resulting possible PPIs are shown on the right.
(D) Retromer structural architecture in different genetic backgrounds. I scores (top right) and P scores (bottom left) are shown for the interactions within the retromer. Each pixel represents a different deletion-bait-prey combination and is the average of 12 independent measurements. Black squares delimit PPIs within the complex in different genotypes. White pixels represent untested or filtered combinations. Numbers in parentheses (above the I score scale) represent the probability of being a PPI. Clustering of the perturbation patterns in homozygous deletion backgrounds reconstructs the subcomplex architecture of the retromer complex (right).
(E) Western blot showing the expression of retromer subunits in different deletion backgrounds.
(F and G) I score of PPIs in wild-type and mutant backgrounds. Bars represent 95% confidence intervals. The dotted line represents the 95% positive PPI threshold.
See also Figure S3.
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6. Figure 4 NPC Structural Organization
(A) PCA screen in wild-type cells. Subunits belonging to the same subcomplex cluster together based on the similarity of their perturbation profiles. Each pixel is the average of seven independent PCA measurements. Only baits or preys showing at least one positive PPI are shown. The full data set can be found in Table S3.
(B) Protein interactomes of the NPC constructed from BioGRID (Stark et al., 2011). Only high-confidence PPIs are shown (reported by at least two different experimental systems). The edge thickness reflects the number of times a PPI has been reported, and edge colors represent the corresponding number of experimental systems. Green nodes represent deletions tested in the perturbation screen. Subcomplexes identified as dense modules in the network are represented on the schematic of the NPC adapted from Alber et al. (2007) and Brohawn et al. (2009). The network was made using Cytoscape (Smoot et al., 2011).
See also Figure S4 and Table S3.
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7. Figure 5 NPC Perturbation
(A) NPC I scores in the wild-type and mutant genetic backgrounds. Each data point is the average of seven measurements of strains constructed independently by SGA and PCA. In the top and right margins are the I score distributions. Significant hits (p < , FDR = 1%) are colored according to their absolute P score value.
(B) Confirmation of the effect of gene deletions on PPIs by spot dilution assays.
(C) Western blot showing the expression of NPC TAP-tagged subunits (Ghaemmaghami et al., 2003) to measure their abundance in different deletion backgrounds for pairs showing tendencies for exclusively negative or positive P scores (from left to right: positive, negative, negative, negative, negative, and positive).
(D) Rank-order distribution of perturbation. NPC gene deletions are ranked as a function of the sum of the absolute P score of their homozygous hits normalized by the total number of PPIs measured in each background. Individual I score plots for nup188Δ and nup170Δ are shown. WT, wild-type.
See also Figure S5 and Table S1.
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8. Figure 6 Robustness of the NPC to Genetic Perturbations
(A) Distribution of GI scores.
(B) P score distributions for the retromer and NPC. Only homozygous significant P scores are considered.
(C) Association of GI scores and P scores. Each data point represents the mean of GIs between the deletion-bait and the deletion-prey pairs, for each deletion-bait-prey combination of the corresponding window of homozygous hits ordered by their P score rank.
(D) P score distributions of homozygous strains (significant P scores) for deletion having paralogs and singletons in the same combination.
(E) Spearman correlation between the ratio of negative GIs and the proportion of paralogous subunits for five different data sets (see Experimental Procedures).
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9. Figure 7
Genetic Robustness of Protein Complexes at the Cellular and Molecular Levels
(A) A cell is robust to the loss of some protein complexes but fragile to the loss of others. Dispensable protein complexes, such as the retromer, are fragile to subunit loss, whereas essential complexes, such as the NPC, are partially robust, mainly because of subunit redundancy (red arrow length).
(B) Gene duplication and other robustness mechanisms affect the assembly landscape of protein complexes by multiplying the number of canalized states. The deletion of a subunit in a fragile protein complex results in its nonassembly (left). Essential protein complexes are robust to genetic perturbations because the deletion of a subunit (red) can be buffered by another one (orange), which in many cases is a paralogous gene. This functional compensation would lead to a relatively stable and functional alternative configuration. In these essential protein complexes, some subunits (black) are essential for cell survival because their deletion cannot be compensated for (right). The depth of the landscape represents the stability of the assembly.
(C) Model for the functional compensation between paralogs (red) at the PPI level. Thick edges represent increased PPIs, and blue ones represent new PPIs.
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10. Figure S1 Detailed Experimental Design, Related to Figure 1
(A) Description of the Protein-fragment Complementation Assay (PCA).
(B) Detailed experimental design. Media composition and description of the design are provided in Extended Experimental Procedures.
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11. Figure S2
Hit Identification and Confirmation for the Retromer, Related to Figure 2
(A) Randomization analysis to determine p value thresholds for three fixed False Discovery Rates (FDR; 5%, 1% and 0.1%). 100 permutations (gray lines) were averaged (black line) to compute the expected FDR. Threshold p values are shown for each of the three FDRs.
(B) Confirmation by spot dilution assay, showing that 14 out of 15 tested hits (>90%) confirmed our large-scale results. MTX: methotrexate containing medium, DMSO: control medium, without methotrexate. Media composition is provided in Extended Experimental Procedures.
(C) Spot dilution assay of positive controls (leucine zipper fused to DHFR fragments) in deletion backgrounds of all retromer subunits, showing no detectable fitness defects on this medium.
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12. Figure S3
Full Heatmap of Retromer Perturbation Screen, Related to Figure 3
Interaction score (I score, top right) and perturbation-score (P score, bottom left) are shown. Each pixel represents a different deletion-bait-prey combination and is the average of 12 independent measurements of independently constructed strains. Black squares delimit PPIs within the complex and with neighboring proteins in different genotypes. White pixels represent untested or filtered combinations. Numbers in parentheses represent probability of being a PPI based on the mixture modeling (see Extended Experimental Procedures). The complete data set can be found in Table S2.
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13. Figure S4
Correlation between Contact Frequency and I Score in the NPC Wild-Type Screen, Related to Figure 4
Red dots represent PCA positive PPIs, black and gray dots PCA negative PPIs. Grey dots are likely to be false negatives in PCA, since these interactions are high confidence consensus PPIs from Alber et al. (2007). False-negatives are expected for instance for cases where the C-termini of the proteins are too distant to allow fragment complementation (Tarassov et al., 2008). Correlation was computed for all data points (gray), or by excluding gray data points (red). Only the filtered data from Alber et al. were used.
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14. Figure S5 NPC Hit Determination and Confirmation, Related to Figure 5
(A) Confirmation by spot dilution assay and independent strain construction, showing that eight out of nine tested combinations confirmed our large-scale results. Fitness in the different NPC deletion backgrounds tested here was also assessed with constitutively interacting artificial constructs (leucine zipper fused to DHFR fragments). L-DHFR: negative control, see Extended Experimental Procedures. MTX: methotrexate containing medium, DMSO: control medium, without methotrexate. Media recipes are provided in Extended Experimental Procedures.
(B) Fitness measurement of all deletion backgrounds on MTX medium. PCA with positive controls (leucine zipper fused to DHFR fragments) was performed in 44 replicates on solid medium and estimated as for the PCA in the perturbation screen. These subtle differences affected colony size estimates in the screen but these effects were eliminated by normalization (see C) such that they do not affect I scores. Bars represent 95% confidence interval.
(C) Comparison of median I score in different deletion backgrounds in the perturbation screen versus colony size of positive controls in the same backgrounds (fitness effects), before (top) and after (bottom) quadrant normalization, showing that the subtle fitness differences observed in (B) were eliminated by the normalization.
(D) Randomization analysis to determine p value thresholds for three fixed FDRs (5%, 1% and 0.1%). 100 permutations (gray lines) were averaged (black line) to compute the expected FDR. Threshold p values are shown for each of the three FDRs.
Cell Reports 2013 3, DOI: ( /j.celrep )
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