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The broad tolerance of domain-rearranging mutations by a yeast signaling network suggests that signaling complexes have loose spatial constraints, making manipulation and perhaps evolution easier., The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering., Author Summary Cells use complex protein interaction networks to sense and process external signals. Proteins involved in signaling are often composed of multiple functional units called domains. Because domains are modular, mutations that rearrange domains among proteins have the potential to result in the creation of novel proteins with altered functions. At an evolutionary timescale, domain rearrangements contribute to the functional diversification of signaling networks; at the shorter timescale of the life of an individual, domain rearrangements can impair cellular functions and lead to disease. Here, we investigated how domain-rearranging mutations alter the function of signaling networks, in particular when these mutations disrupt pre-existing proteins. We used as a model system the yeast mating signaling pathway, which shares many properties with more complex pathways active in human cells. Our results demonstrate that signaling networks are often robust to domain rearrangements that disrupt pre-existing genes. In addition, our experiments suggest a possible mechanism to explain this robustness: rather than being a rigid multi-protein machine, the yeast mating signaling complex is a dynamic ensemble with loose spatial constraints. Because of this, the changes in protein interaction partners caused by domain-rearrangement mutations can be accommodated without disrupting network function.