Stabilization of HSA32, an aggregation-prone protein, by the protein disaggregase HSP101 plays a critical role in maintaining acquired thermotolerance in Arabidopsis

Abstract

Heat acclimation confers acquired thermotolerance (AT), and heat-acclimation memory (HAM) is the maintenance of AT for some time. In Arabidopsis and rice, the heat-stress-associated 32-kDa protein (HSA32) and the protein disaggregase heat shock protein101 (HSP101) form a positive feedback loop at the protein level to maintain AT; HSA32 mitigates HSP101 degradation, and HSP101 positively regulates the accumulation of HSA32. Here, we report the underpinning mechanism regarding how HSP101 affects the HSA32 level in Arabidopsis. We found that, without HSP101, nascent HSA32 was rapidly degraded, and the proteasome inhibitor, bortezomib, inhibited the degradation. In response to heat stress, the nascent HSA32-GFP fusion protein was present in liquid condensates and diffused in the cytosol after returning to non-stress temperature. Proximity labeling with HSA32-TurboID identified HSP101 and five other protein chaperones and co-chaperones as the primary interactors. Disturbing the interaction between HSA32 and HSP101 destabilized HSA32 and compromised HAM. HSA32 is predicted as a TIM-barrel protein with three intrinsically disordered regions of high aggregation propensity. Recombinant HSA32 expressed in E. coli was partitioned into insoluble fractions, suggesting that HSA32 is aggregation-prone. Our findings highlight how the interplay between an aggregation-prone protein and a protein disaggregase can maintain plant stress memory.