Figure 1: Figure 1A shows the possible fates of a pair of paralogs; they can either become functionally independent, retain some redundant elements, or become functionally dependent on each other. Duplication of a gene also affects its protein-protein interaction network, as the paralogs can independently lose or gain ability to interact with proteins. As seen in Figure 1B, when one paralog is deleted, compensatory activity presents as an increase in PPI intensity and dependent activity presents as a decrease in PPI. PPI was measured using a protein-fragment complementation assay; fragments of an enzyme necessary for growth in a restrictive medium are fused to proteins so protein interaction intensity can be measured as a function of colony growth. Figure 1C graphs PPI intensity in wild-type vs. paralog-deleted conditions. Interaction scores were strongly correlated, indicating that generally paralog deletion does not significantly affect PPI network, but several genes deviated from the best fit line, indicating significant compensatory or dependent relationships. Figure 1D shows that compensation and dependency typically did not occur in the same paralog pair.

Figure 2: Only a few cases showed a significant increase in protein levels via flow cytometry in the paralog-deleted condition (Figure 2A), which does not support paralog up-regulation as a mechanism. Figure 2B shows an alternative mechanism for compensation, where the paralogs are mutually exclusive; one paralog shows stronger interaction in the wild-type case because it is more abundant or has a higher affinity. If it is deleted, the other paralog is able to compensate. They tested this theory buy overexpressing the compensating paralog, expecting a decrease in the interaction of the originally dominant paralog. Figure 2C shows a case where this hypothesis was true; the interaction score of the protein in red decreased when its compensating paralog was overexpressed. Figure 2D shows a case where this hypothesis was not true; the interaction score of the protein in red was not affected by overexpressing of its paralog. Overall, paralogs pairs classified as "compensating" were more likely to show decreased PPI upon overexpression of the compensating paralog (Figure 2E)

Figure 3: Figure 3 examines dependent paralog pairs. Figure 3A shows that dependent paralog pairs are significantly more likely to be heteromers, or two proteins that physically interact. Figure 3B shows that in asymmetrical dependent pairs, where one protein can function without the other but not vice versa, the independent protein tends to have a higher abundance. The dependent protein abundance tends to lower further when its independent counterpart is deleted, as measured by flow cytometry and/or Western blots (Figure 3C). Figure 3D shows how different types of gene duplication affect how deletion of one paralog impacts fitness. Deletion of heteromer that originated from small-scale duplications was significantly more detrimental than deletion of singletons, or a non-duplicated gene, while deletion of ohnologs was less detrimental than deletion of singletons. In both cases, deletion of paralogs that formed heteromers had a larger negative impact on fitness than their non-heteromer counterparts, which supports that heteromers are working as functional units; if one is deleted, the other is unable to function.

Figure 4: Figure 4A illustrates three possible dimer configurations after whole genome duplication. Figure 4B illustrates the method of determining the ancestral origin of a current paralogous heteromer resulting from whole genome duplication (ohnomer), comparing the current paralog pair to its ortholog in a related species that did not undergo whole genome duplication. This method revealed that paralogous heteromers were more likely to have a homomer ortholog (Figure 4C), supporting the mechanism outlined in Figure 4A. Additionally, ohnologs that originated from a protein that formed homodimers are more likely to retain both paralogs (Figure 4D), further supporting functional dependence of this class of proteins. In this configuration, the dependent duplicate tends to accumulate more nonsynonymous mutations than its independent counterpart (Figure 4E), suggesting that the presence of an independent paralog allows for greater mutational freedom. Figure 4F summarizes the entire paper: paralogs pairs that originated from ancestral homologs become functionally dependent heteromers after duplication, causing loss of function if one paralog is deleted.

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