RT Journal Article
SR Electronic
T1 Evaluating Client Protein Recovery by the Hsp40s DNAJB8 and DNAJB1 with AP-MS
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.03.31.485989
DO 10.1101/2022.03.31.485989
A1 Montoya, Maureen R.
A1 Quanrud, Guy M.
A1 Mei, Liangyong
A1 Montaño, José L.
A1 Genereux, Joseph C.
YR 2022
UL http://biorxiv.org/content/early/2022/03/31/2022.03.31.485989.abstract
AB Hsp40s play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. These misfolded client proteins are then delivered to Hsp70. Mutation of the Hsp40 J-domain blocks Hsp70 binding, inhibiting client protein release from Hsp40. We previously integrated misfolded protein recognition by Hsp40 into a platform to identify proteins that are destabilized by cellular stress. However, the dependences of Hsp40 interactions and client recovery on J-domain activity, Hsp40 identity, and crosslinking have not been addressed. Herein, we apply quantitative proteomics to systematically characterize the interactions networks of human Hsp40s DNAJB8 and DNAJB1 with intact or inactivated J-domains. We find that DNAJB8 irreversibly binds over a thousand protein interactors even in the absence of stress. Inactivation of the J-domain decreases interaction with Hsp70 family and associated proteins, but does not generally affect client binding. By contrast, J-domain inactivation and cellular crosslinking substantially increase the relative recovery of proteins from DNAJB1 co-immunoprecipitation. This advantage is completely offset by loss of DNAJB1 recovery under these conditions, making DNAJB1 a poor bait for client protein recovery as compared to DNAJB8. The J-domain inactivated DNAJB8H31Q has increased affinity to its client proteins under heat stress, while no such change in affinity is observed for the wild-type protein, despite their similar client binding profiles under basal conditions. Hence, we find that DNAJB8H31Q is an effective recognition element for the recovery of destabilized client proteins following cellular stress. Raw mass spectrometry data is accessible through the PRIDE archive (pxd030633).Competing Interest StatementThe authors have declared no competing interest.