Subtle Variations in a Client Protein Determine Bacterial Hsp90 Dependence

Abstract

Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilS_So) in the bacterium Shewanella oneidensis. Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilS_So from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli, suggesting that E. coli TilS (TilS_Ec) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify determinants of Hsp90 dependence. We found that in contrast to TilS_So, TilS_Ec was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilS_Ec is not a client of Hsp90. Chimeras between TilS_So and TilS_Ec as well as directed mutagenesis revealed a region of TilS_So that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilS_Ec was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilS_So instead of TilS_Ec. Altogether, our work reveals that protein-specific features, such as stability and degradation sensitivity, can determine whether orthologous proteins require the bacterial Hsp90 chaperone in vivo.