Graphical Abstract Highlights d SDS22 and I3 form a transient inactive complex with PP1 during PP1 biogenesis d SDS22-PP1-I3 disassembly by the p97 AAA-ATPase allows holoenzyme formation d Direct binding of I3 by the SEP domain of the p37 adapter mediates p97 recruitment d Disassembly involves ATP-driven pulling of I3 into the channel of the p97 hexamer Correspondence hemmo.meyer@uni-due.de In BriefWeith et al. demonstrate that newly synthesized PP1 is first held in an inactive complex by SDS22 and inhibitor-3 that needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Disassembly involves direct interaction of the p37 adapter with inhibitor-3, followed by its ATP-driven unfolding. SUMMARYThe functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes.Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange. and helped with supervision of the project. J.H. generated adapter cell lines. I.P. and A.M. generated and provided baculoviruses coding for PP1 components. F.K. and M. Kaiser performed mass spectrometry measurements. J.d.P.G. and M.B. advised and analyzed data. H.M. conceived and supervised the project and wrote the manuscript. DECLARATION OF INTERESTSThe authors declare no competing interests.

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