RPAP3

RPAP3 is the central scaffolding subunit of the R2TP co-chaperone complex, coupling the HSP90 chaperone machinery to the RUVBL1/RUVBL2 AAA-ATPase ring to drive the assembly of macromolecular complexes (PMID:29662061). Its conserved C-terminal domain directly contacts the ATPase domain of RUVBL2 to nucleate R2TP assembly, while its mobile TPR domains project to the opposite face of the RUVBL ring and associate with PIH1D1, which mediates client recruitment; RPAP3 thus spans both faces of a single RUVBL hexamer as an extended scaffold and flexible tether for HSP90 (PMID:29662061). The TPR domains engage HSP90 specifically through its C-terminal EEVD/MEEVD motif via a two-carboxylate clamp, discriminating against the HSP70 VEEVD motif, and this engagement modulates HSP90 ATPase activity (PMID:18412542, PMID:24012479). Through PIH1D1 binding the same protein region stabilizes the otherwise labile PIH1D1 partner (PMID:22179618, PMID:23159623). This architecture supports quaternary protein folding of diverse clients, exemplified by the testis-enriched RPAP3-like paralog SPAG1, which forms an analogous R2SP complex required for liprin-α2 assembly (PMID:29844425), and by client classes including snoRNP core proteins and RNA polymerase II in the orthologous machinery (PMID:24394412). RPAP3 function is regulated by CK2 phosphorylation at Ser116/Ser119/Ser121, where the unphosphorylated form preferentially binds ribosomal preassembly complexes, linking RPAP3 to ribosome biogenesis (PMID:35129352). Beyond core chaperone scaffolding, RPAP3 sequesters the miRNA-pathway factor TRBP through a TPR1–dsRBD3 interaction that is mutually exclusive with TRBP–Dicer binding, thereby modulating miRNA biogenesis (PMID:35150569), and it participates in the UV-induced DNA damage response together with RUVBL2 via regulation of H2AX phosphorylation (PMID:19180575).

1. 2008 Medium

   Established the molecular basis by which RPAP3/Tah1 selects HSP90 over HSP70, defining the chaperone connection at the heart of R2TP.

   Evidence Binding assays and mutagenesis with yeast/human HSP90 and HSP70 plus ATPase readout in yeast Tah1

   PMID:18412542

   Open questions at the time

   • Functional consequence of HSP90 ATPase modulation for client folding not resolved
   • Did not address how Tah1 couples to the RUVBL ring
2. 2010 High

   Showed that heterodimerization with PIH1 reshapes RPAP3/Tah1's effect on HSP90, converting ATPase stimulation into inhibition and revealing complex-dependent regulation.

   Evidence Analytical ultracentrifugation, microcalorimetry, native mass spectrometry, and ATPase assays on the yeast Pih1-Tah1 heterodimer

   PMID:20663878

   Open questions at the time

   • Physiological clients whose folding depends on this ATPase tuning not identified
   • Human RPAP3-PIH1D1 ATPase regulation not directly tested
3. 2009 Medium

   Linked RPAP3 to the UV DNA damage response by tying it to RUVBL2 and H2AX phosphorylation, extending its role beyond chaperone scaffolding.

   Evidence AP-MS, co-IP, confocal microscopy, and RNAi with UV survival/H2AX readouts in HeLa cells

   PMID:19180575

   Open questions at the time

   • Direct molecular mechanism connecting RPAP3 to H2AX kinases unresolved
   • Whether the effect requires intact R2TP not tested
4. 2011 High

   Provided the first atomic view of RPAP3/Tah1's TPR architecture and showed how its disordered C-terminal region stabilizes the labile PIH1 partner.

   Evidence NMR structure of yeast Tah1 with binding and in vitro/in vivo stability assays

   PMID:22179618

   Open questions at the time

   • Structure of the full R2TP assembly not yet defined
   • Client engagement mechanism not addressed
5. 2013 High

   Defined the residue-level carboxylate clamp and capping helix that recognize the HSP90 MEEVD motif, pinpointing the recognition determinants.

   Evidence High-resolution NMR structures of free and HSP90-peptide-bound Tah1 with mutagenesis

   PMID:24012479

   Open questions at the time

   • How HSP90 tethering is coupled to RUVBL/PIH1 client handoff not shown
6. 2014 Medium

   Demonstrated that the RPAP3 ortholog bridges HSP70 and HSP90 and is required for assembly of snoRNPs, TOR signaling, and RNA polymerase II, framing R2TP as a client-handoff machine.

   Evidence Co-IP, RNAi/null mutants, and chaperone activity assays in Drosophila Spag

   PMID:24394412

   Open questions at the time

   • Direct human RPAP3-HSP70 interaction not structurally defined
   • Order of HSP70-to-HSP90 client transfer inferred, not demonstrated
7. 2018 High

   Resolved how RPAP3 organizes the human R2TP complex, showing its C-terminal domain docks on RUVBL2 while TPR/PIH1D1 face the opposite ring side, and that a paralog (SPAG1) builds an analogous R2SP complex for tissue-specific client folding.

   Evidence Cryo-EM (3.6 Å), structural and biochemical mapping, and functional knockdown across two studies

   PMID:29662061 PMID:29844425

   Open questions at the time

   • Conformational dynamics during active client folding not captured
   • Full set of human R2TP clients incomplete
8. 2022 High

   Identified two regulatory layers: CK2 phosphorylation gating RPAP3's binding to ribosome biogenesis factors, and a structurally defined RPAP3-TPR1–TRBP interaction that competes with Dicer to modulate miRNA biogenesis.

   Evidence AP-MS, in vitro kinase and phospho-null mutagenesis; plus 1.5 Å crystal structure with competitive binding and HSP90 inhibition

   PMID:35129352 PMID:35150569

   Open questions at the time

   • Physiological signals triggering CK2 control of RPAP3 not defined
   • Cellular consequences of TRBP sequestration for specific miRNAs not mapped

• How RPAP3's distinct activities—R2TP scaffolding, DNA damage response, miRNA regulation, and NF-κB modulation—are integrated or selected within a single cell remains unresolved.
• No unifying model linking chaperone scaffolding to signaling and stress roles
• Whether non-R2TP functions require RUVBL/PIH1D1 or HSP90 is untested