HOP is a monomer: Investigation of the oligomeric state of the co‐chaperone HOP

Abstract

The co-chaperone Hsp70-Hsp90 organizing protein (HOP) plays a central role in protein folding in vivo, binding to both Hsp70 and Hsp90 and bringing them together in a functional complex. Reports in the literature concerning the oligomeric state of HOP have been inconsistent-is it a monomer, dimer, or higher order oligomer? Knowing the oligomeric state of HOP is important, because it places limits on the number and types of multiprotein complexes that can form during the folding cycle. Thus, the number of feasible models is simplified. Here, we explicitly investigate the oligomeric state of HOP using three complementary methods: gel filtration chromatography, sedimentation equilibrium analytical ultracentrifugation (AUC), and an in vivo coexpression assay. We find that HOP does not behave like a monomeric globular protein on gel filtration. Rather its behavior is consistent with it being either an elongated monomer or a dimer. We follow-up on these studies using sedimentation equilibrium AUC, which separates on the basis of molecular weight (MW), independent of shape. Sedimentation equilibrium AUC clearly shows that HOP is a monomer, with no indication of higher MW species. Finally, we use an in vivo coexpression assay that also supports the conclusion that HOP is a monomer.

Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop

“…4b). Release of the TPR1-DP1 module from the Hsp90:Hop complex core could allow Hsp90 and Hop interaction with other chaperones or co-chaperones, in accordance with other studies 14,27 . The arrangement of the Hop domains in the complex seems to prevent total closure of the Hsp90 dimer, thus blocking ATPase activity and generating a semiclosed conformation that allows client protein binding 16,27,28 .…”

“…This domain is located in the Hsp90 monomer, opposite the monomer that interacts with the Hop TPR2A-TPR2B-DP2 module; this supports the idea of asymmetric interaction of the Hsp90 dimer with other chaperones and cochaperones 14,16,27 . Once the interaction between Hsp70 and Hop takes place, there appears to be a further interaction between Hsp70 NBD and the Hsp90-N on one Hsp90 monomer, which Fig.…”

“…These processes are aided by various co-chaperones 12 , one of the best studied of which is the Hsp70-Hsp90-organizing protein (Hop; Sti1 in yeast) 13 , a monomer 14 composed of three tetratricopeptide repeat domains (TPR1, TPR2A, TPR2B) involved in protein-protein interaction, and two small aspartic acid-proline repeat domains (DP1, DP2) involved in client activation 15,16 ( Supplementary Fig. 1c).…”

Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

“…Initially, we found that Hsp90 was predominantly dimeric and FKBP52 monomeric, both were anticipated. Recently there has been some controversy over the oligomeric state of Hop reported as dimeric (6,20,34) or monomeric (22,35) and Hsp70 can form high oligomeric species under some conditions (36). We have shown that under the conditions used here both proteins are monomeric.…”

Aging induces a distinct gene expression program in mouse islets

“…stress-induced phosphoprotein 1; Hsp70-Hsp90 organizing protein; Hop; STI1; p60) is a cochaperone protein that has been implicated with protein folding. 42 Stip1 has been indirectly implicated in b-cells. Pdx1 is known to occupy a site near Stip1.…”