Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation

Abstract

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.

SDS22 selectively recognizes and traps metal-deficient inactive PP1

“…SDS22 is a highly conserved PP1binding protein, but its function in PP1 maturation and regulation is not well understood. SDS22 has been described as a PP1 inhibitor, a PP1-specific chaperone, and a PP1 nuclear targeting protein (20,24,29,30). These contradictory models of SDS22 function have posed a long-standing conundrum.…”

“…In mammalian and yeast cells, SDS22 and I-3/Ypi1 associate with newly synthesized PP1/Glc7 and together are hypothesized to play an essential role in PP1 biogenesis (28,30). Furthermore, defects in yeast Sds22 and I-3/Ypi1 result in aggregation of newly synthesized PP1/Glc7 (29).…”

“…Loss of Sds22 function leads to Glc7 aggregation, which leads to the hypothesis that Sds22 with I-3 (Ypi1 in S. cerevisiae) prevents Glc7 misfolding (26)(27)(28)(29). Finally, SDS22, which is highly expressed in cells, was recently hypothesized to function as a PP1 maturation factor via an unknown mechanism and, thus, is critical for PP1 biogenesis (30). Taken together, the Significance PP1, a metal-dependent enzyme responsible for >50% of all dephosphorylation reactions, forms distinct holoenzymes with >200 regulatory proteins, which create specificity for its substrates.…”

Structure of the PUB Domain from Ubiquitin Regulatory X Domain Protein 1 (UBXD1) and Its Interaction with the p97 AAA+ ATPase

“…p97 comprises a regulatory N-terminal domain, two AAA ATPase domains (D1 and D2) that form a stacked hexamer, and a short, disordered C-terminal region [4,5]. Protein unfolding is mediated by ATP-driven translocation of substrate proteins through the central channel of the hexamer [6][7][8][9]. The function and activity of p97 is regulated by a large number of cofactor proteins [10].…”

“…The function and activity of p97 is regulated by a large number of cofactor proteins [10]. They include substrate adapters that bind at the N-domain and assist insertion of the substrates into the D1 pore [9,11] and also many regulatory proteins that modulate p97 activity in different cellular processes [10].…”