Abstract
Cell polarity is a near universal trait of life and guides many aspects of animal development. Although a number of key polarity proteins have been identified, many interactions with proteins acting downstream likely remain to be elucidated. Mutations in polarity proteins or deregulation of polarity pathways are causative for various
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diseases including cancer. To identify new proteins or new links between polarity regulators, we combined a fragment based high-throughput yeast two-hybrid approach with in-vivo RNAi studies using existing models of polarity in the small nematode Caenorhabditis elegans. In total we identified 459 protein interactions between 296 proteins with Y2H, which greatly expand the polarity interaction network. Computational and experimental approaches were used to validate the quality of our network. The polarity interactome was characterized in several C. elegans polarity marker strains by RNAi. This led to 100 protein pairs that physically interact and for which RNAi of the corresponding genes causes a defect in the same polarity-related process. One interaction was investigated in more detail, the interaction between the RhoGAP protein PAC-1 and the polarity protein PAR-6. Our experiments show that PAC-1 lacking the PAR-6 binding domain expressed in a pac-1 mutant is unable to rescue the PAR-6 localization defect, even though mutant PAC-1 localizes as wild-type. The network of candidate polarity-related interactions we identified will be a valuable resource for the research community. We expanded the concept of Y2H fragment based interaction mapping to a human genome wide scale. Protein function is mostly dictated by its structure and by its interactions. Full-length proteins are often not capable to perform in screening methods because of e.g. miss-folding. We generated a fragment based human Y2H prey library by mechanically fragmenting the ORFeome 5.1 library. The new library was experimentally validated by screening 44 bait proteins involved in cell polarity and cell division with well described interactions. We identified 22 protein interactions of which 8 were described previously. Furthermore, we are able to identify new minimal interaction domains. These 22 interactions were validated by protein affinity purification and 12 (55%) tested positive. Moreover, we validated the newly identified minimal interaction domains for these 12 protein pairs by affinity purification. We identified with Y2H the C. elegans protein HMGS-1 (HMG-CoA synthase), the key regulator of the Mevalonate pathway, interacting with the small ubiquitin modifier protease ULP-4. HMGS-1 is the first step of the Mevalonate pathway which facilitates the syntheses of steroid hormones and cholesterol precursors. Perturbations of this pathway are causative to cardiovascular diseases and cancer. During the lifespan of C. elegans the expression and localisation pattern of ULP-4 changes from cytoplasmic in young animals to localizing to mitochondria in old animals. When ULP-4 is in the cytoplasm, the protein sumoylates HMGS-1, by which metabolism of the worm increases. When worms age, ULP-4 is relocated to the mitochondria, upon which HMGS-1 will be desumoylated and degraded by the proteasome, decreasing metabolism specifically altering the Mevalonate pathway. This conserved regulatory rewiring mechanism may be a reference for future therapies.
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