ABSTRACT
Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Protein Kinases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Enzyme Stability , Gene Expression Regulation, Fungal , HSP90 Heat-Shock Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Chaperones/genetics , Multiprotein Complexes , Protein Binding , Protein Kinases/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Signal Transduction , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolismABSTRACT
Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis. Here we report several principles underlying the assembly and topological organization of the highly conserved SAGA and NuA4 co-activator complexes, which share the Tra1 subunit. We show that Tra1 contributes to the overall integrity of NuA4, whereas, within SAGA, it specifically controls the incorporation of the de-ubiquitination module (DUB), as part of an ordered assembly pathway. Biochemical and functional analyses reveal the mechanism by which Tra1 specifically interacts with either SAGA or NuA4. Finally, we demonstrate that Hsp90 and its cochaperone TTT promote Tra1 de novo incorporation into both complexes, indicating that Tra1, the sole pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the assembly of transcriptional complexes and reveals the contribution of dedicated chaperones to this process.
