Abstract
Many proteins function as part of multimeric complexes rather than individually. These complexes regulate many cellular functions such as metabolism, gene expression or DNA damage repair. All these pathways are regulated by at least one member of the PIKK family of kinases. So far, although many efforts have been made to characterize the biochemical composition and stoichiometry of PIKK-containing complexes, our knowledge of the dynamics and regulation of their assembly remains scarce. Cells need to coordinate the expression, maturation, and assembly of individual subunits because defects in these processes result in the accumulation of misfolded or unassembled subunits, which compromises their functions and can cause proteotoxic stress. My thesis project aimed at identifying the principles underlying the assembly of large multifunctional complexes, using the SAGA co-activators as a paradigm. First, I implemented a genetic tool, called Recombination Induced Tag Exchange (RITE), in the fission yeast Schizosaccharomyces pombe. This tool allows a controlled epitope switch within a protein of interest and I used it to purify newly synthetized SAGA complexes. Unfortunately, this tool does not have sensitive and resolutive enough to identify assembly intermediates or specific assembly factors. However, I derived the RITE system to follow the fate of newly synthetized Tra1, a SAGA subunit and revealed that SAGA assembly follows an ordered pathway. Indeed, the Spt20 subunit is necessary and sufficient to recruit Tra1, which itself causes the de novo incorporation of the DUB module. Next, we provided new insights into the mechanism by which the Hsp90 cochaperone, TTT, stimulates PIKK maturation and assembly. First, I showed that TTT and Hsp90 are required for the de novo incorporation of Tra1 into SAGA. Second, I demonstrated that TTT binds to its substrates cotranslationally, by recognizing the most conserved domain of PIKKs. Together with other results from the laboratory, our work suggests that there is a spatial and temporal segregation between the acquisition of the native form of PIKKs and their assembly. The role of TTT would therefore be to chaperone PIKK during synthesis in order to protect nascent polypeptides from degradation, aggregation or premature interactions, while synthesis completes. Overall, my work provides new insights into the molecular basis of multifunctional complex assembly, which is important to understand their structural organization and their regulation.