A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins

Abstract

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins

“…It makes intuitive sense, however, that the septin NC interface, which at least for Cdc11 and Shs1 is exposed at both ends of a septin octamer, would not attract much cytosolic chaperone attention. A recent study that crosslinked Lys residues (Nitika et al, 2021) found a direct interaction between Ssa1 and a Lys (478) in the CTE of Cdc3, far from the G and NC interfaces (Supplemental Figure 9) and within the putative coiled coil formed in the non-native G Cdc3 homodimer (Figure 1). These crosslinks with Ssa1 probably only occurred because the other Ssa chaperones were eliminated (Nitika et al, 2021), otherwise Ssa2, Ssa3 or Ssa4 would presumably have "outcompeted" Ssa1 for septin interaction.…”

“…A recent study that crosslinked Lys residues (Nitika et al, 2021) found a direct interaction between Ssa1 and a Lys (478) in the CTE of Cdc3, far from the G and NC interfaces (Supplemental Figure 9) and within the putative coiled coil formed in the non-native G Cdc3 homodimer (Figure 1). These crosslinks with Ssa1 probably only occurred because the other Ssa chaperones were eliminated (Nitika et al, 2021), otherwise Ssa2, Ssa3 or Ssa4 would presumably have "outcompeted" Ssa1 for septin interaction. As the crosslinked lysine in Ssa1 lies on the surface of the lid of the substrate-binding domain, it is possible that, once the Cdc3 NTE is displaced by interaction with Cdc12, the Ssa1 substrate-binding pocket does engage the Cdc3 G interface, bringing the Lys-rich Cdc3 CTE near the Ssa1 lid (Supplemental Figure 9).…”

The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

“…A challenge in understanding the differential role of the Ssa paralogs is a lack of verified client proteins. While several Hsp70 interactomes have been published under differing conditions and phosphorylation site mutations, validation of these and their cellular effect is still under investigation [33, 43–47]. Excellent attempts to dissect the roles of Ssa paralogs include the well-established Hsp90 client (Ste11) and a non-yeast client, v-Src in addition to the yeast prions [ URE3 ] and [ PSI + ] [21].…”

The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

“…A challenge in understanding the differential role of the Ssa paralogs is a lack of verified client proteins. While several Hsp70 interactomes have been published under differing conditions and phosphorylation site mutations, validation of these and their cellular effect is still under investigation [ 37 , 47 – 51 ]. Excellent attempts to dissect the roles of Ssa paralogs include the well-established Hsp90 client (Ste11) and a non-yeast client, v-Src in addition to the yeast prions [ URE3 ] and [ PSI + ] [ 23 , 25 , 52 , 53 ].…”