Extended Linkers Improve the Detection of Protein-protein Interactions (PPIs) by Dihydrofolate Reductase Protein-fragment Complementation Assay (DHFR PCA) in Living Cells

Abstract

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.

Fig. 1.

Longer linkers increase signal-to-noise ratio in large-scale Protein-fragment complementation assay (PCA) screens and allow the detection of new PPIs. A, PPIs Z_s (representing a quantitative deviation from the background noise) obtained in a large-scale screen using baits fused to the DHFR F[1,2] fragment with a 3xL (left) and a 4xL (right) compared with a 2xL. PPIs with a significant difference are highlighted with red triangles (3xL) and squares (4xL). B, Distribution of colony size in the DHFR PCA assay with the 2xL and 4xL. 13 baits were screened against the DHFR F[3] yeast collection (n = 3600) with both the 2xL and 4xL (26 baits total). The distribution of normalized colony sizes (interaction score, I_s) is shown for a set of computationally identified most likely true negative and positive interaction partners, TN and TP respectively. The two modes of the bimodal distribution of colony sizes for the TP represent PPIs that are detected (highest mode) and those that cannot be distinguished from the background growth (lowest mode). The signal-to-noise ratio, illustrated as the difference between the medians of the TP over the TN, increases with the 4xL. C, Precision and number of detected PPIs as a function of Z_s thresholds. Precision, or fraction of TP, was calculated as the ratio of TP over all positives (TP + FP) at various values of Z_s for the 2xL and 4xL. A conservative threshold corresponding to a Z_s of 3 is indicated by a dotted vertical line on the graph. D, Retromer PPI network. Colored nodes: retromer subunits. Blue nodes: Vps35 interaction partners. Black lines: all known PPIs between Vps35 and its interaction partners and among these interaction partners. Red lines: newly detected PPIs with Vps35 in the large-scale experiment with the 4xL and confirmed in small scale experiments.

Fig. 2.

PCA signal increases using a longer linker. Detected PPIs after data filtering for the intra complex PCA experiment. Blue circles: RNApol I, II, and III. Orange squares: proteasome. Purple triangles: COG complex. Empty shapes: quantitatively changed PPIs (significantly decreased or increased when compared with 2xL-2xL reference interactions). Solid shapes: new PPIs (PPI not detected with the 2xL-2xL reference linker but detected with a longer linker combination). The p-values are from FDR-corrected t-tests on colony sizes.

Fig. 3.

The use of a longer linker proves to be useful to infer the super-organization of protein complexes. A, Proportions of quantitatively changed PPIs and new PPIs versus unchanged PPIs for all complexes considering every reciprocal interactions such as X-DHFR F[1,2]-Y-DHFR F[3] and Y-DHFR F[1,2]-X-DHFR F[3] as a single PPI. B, Circle plots of all detected PPIs for each complex. Line thickness is proportional to the difference between the 4xL-4xL and 2xL-2xL PCA signal for each PPI. Gray lines: unchanged PPIs. Green lines: negatively changed PPIs. Pink lines: positively changed and new PPIs. Stripe patterns inside the colored boxes represent proteins that were absent from the experiment. C, Proportion of PPIs detected for each combination of subcomplexes within complexes.

Fig. 4.

Longer linkers allow for the detection of PPIs among more distant proteins within complexes. A, Structures of the RNApol I, II, and III and of the proteasome. Green: proteins shared by at least two out of the three RNApol. Dark red: proteasome catalytic subunit. Red: proteasome base. Orange: proteasome lid. Proteins located at different distances or in different subunits are highlighted on each structure. Examples of distances between C-termini of these proteins and their associated PPI Z_s. PPIs detected with longer linkers but not with the 2xL are indicated in the tables. DHFR fragments have also been modeled and are presented at the same scale as the proteasome structure. B, (Left) Correlation between all of the detected PPIs in the proteasome (Z_s) and the distance between the C-termini (2xL-2xL: Spearman r = -0.34, p-value = 2.249e-15; 2xL-4xL: r = -0.36, p-value < 2.2e-16; 4xL-2xL: r = -0.36, p-value < 2.2e-16; 4xL-4xL: r = -0.40, p-value < 2.2e-16). Data were binned into 10 classes for representation only, the correlation tests were performed on raw data. (Right) Distribution of cumulative Z_s for the proteasome PPIs as a function of protein pairwise distances. C, Pairwise C-termini distances for the PPI that are not affected by linker length, those that have enhanced interaction score (I_s) and those that were not detected with the 2xL but are detectable with the 4xL. Data from the RNApol and proteasome complexes are combined in these analyses. p-values of Wilcoxon tests are shown.