A Systematic Approach for the Genetic Dissection of Protein Complexes in Living Cells

•Systems genetics strategy for the perturbation of protein interactomes•Insights into the architecture of the retromer and the nuclear pore complex•The robustness of the nuclear pore complex is due to compensating paralogs•Paralogous proteins modify the assembly landscape of protein complexes

SummaryCells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. One major challenge in cell biology is to elucidate the organization and mechanisms of robustness of these complexes in vivo. We developed a systematic approach to study structural dependencies within complexes in living cells by deleting subunits and measuring pairwise interactions among other components. We used our methodology to perturb two conserved eukaryotic complexes: the retromer and the nuclear pore complex. Our results identify subunits that are critical for the assembly of these complexes, reveal their structural architecture, and uncover mechanisms by which protein interactions are modulated. Our results also show that paralogous proteins play a key role in the robustness of protein complexes and shape their assembly landscape. Our approach paves the way for studying the response of protein interactomes to mutations and enhances our understanding of genotype-phenotype maps.

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