Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants

Abstract

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The ‘T’ component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

The structure of the R2T complex reveals a different architecture of the related HSP90 co-chaperones R2T and R2TP

“…To determine whether the interaction mechanism of RPAP3 and HSP90 is conserved in plants, we modeled the structure of the TPR domain of AtRPAP3 bound to the MEEVD peptide based on the structure of the peptide bound to human RPAP3 TPR2 (Henri et al, 2018), as we could not detect the TPR of AtRPAP3 in the cryoEM analysis because it is located in a region with high flexibility. Modeling showed that the AtRPAP3 TPR can adopt a 3D structure similar to that of human RPAP3 TPR2 (Figure 7A), as shown for the TPR domain of sorghum RPAP3 (Machado Antonio et al, 2023). Alignment of RPAP3 sequences from humans and Arabidopsis showed that the six residues of human TPR2 that contact HSP90 (Henri et al, 2018) are conserved in AtRPAP3 TPR (Supplementary Figure 4; marked with red asterisks).…”

“…One of the functions of RPAP3 within the R2TP complex in animals and yeast is the recruitment of the chaperone HSP90 via the interaction between the conserved Cterminal end of the chaperone and the TPR domain of RPAP3 (Pal et al, 2014). The TPR domain of AtRPAP3 shows significant homology with human RPAP3 TPR2 that specifically interacts with HSP90 (Supplemental Figure 4), as observed for sorghum SbRPAP3 (Machado Antonio et al, 2023). We found that AtRPAP3 was able to interact with AtHSP90.3, one of the four highly homologous cytosolic HSP90 isoforms in Arabidopsis (Krishna and Gloor, 2001), by Y2H (Figure 2 and Supplemental Figure 3), suggesting that the role of RPAP3 in the recruitment of HSP90 is conserved in plants.…”

“…Similarly, in vitro pull-down assays showed that sorghum SbRPAP3 can form a complex with human RuvBL1 and RuvBL2 (Machado Antonio et al, 2023).…”

“…In humans, Tah1 ortholog RPAP3 is a larger protein (75 KDa) containing two TPR domains and a specialized C-terminal domain named RuvBL2-Binding Domain (RBD) for being involved in the interaction with RuvBL2 (Jeronimo et al, 2007;Martino et al, 2018). The plant RPAP3 ortholog (44 KDa) containing a single TPR domain and the RBD has been identified in sorghum (Figure 1A) (Machado Antonio et al, 2023). Pih1 and its ortholog in animals PIH1D1 contain a C-terminal CS domain that mediates interaction with Tah1/RPAP3 and an N-terminal PIH domain that interacts with a specific phosphorylated motif in client proteins (Horejsi et al, 2014).…”

Trends and Prospects of Genetic and Molecular Research in Plants

“…This phenomenon emphasized the importance of HDAC inhibitors in the culture process of calluses. Antonio et al [ 8 ] cloned a putative gene corresponding to RPAP3, which plays the same role as the chaperone R2TP, in the monocot Sorghum bicolor , characterizing the SbRPAP3 protein with 396 residues. As a result, SbRPAP3 not only maintained its functions, including binding to RUVBL, Hsp90, and Hsp70, but also showed that RPAP3 performs an important function in the R2TP complex.…”