Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein's propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.
Environmental stresses such as heat cause generation of misfolded and damaged proteins, which are highly toxic and must be efficiently removed. In plants, NBR1, the first isolated autophagy receptor with an ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting ubiquitinated protein aggregates under stress conditions for degradation by autophagy. To study how stress-induced misfolded and damaged proteins are detected and ubiquitinated in plant cells, we analyzed the chaperone-associated E3 ubiquitin ligase CHIP from Arabidopsis thaliana for its role in protection against proteotoxicity in plant stress responses. Disruption of Arabidopsis CHIP caused increased sensitivity to a spectrum of abiotic stresses as found in the Arabidopsis nbr1 mutants. Disruption of both Arabidopsis CHIP and NBR1 further compromised plant stress tolerance, indicating that their roles are additive. Furthermore, in the chip nbr1 double mutant, compromised heat tolerance was associated with increased accumulation of insoluble proteins derived mostly from heat-sensitive but biologically important proteins such as Rubisco activase, catalases and proteins required for protein synthesis and folding. Importantly, stress-induced protein aggregates were still highly ubiquitinated in the chip mutants. These results strongly suggest that CHIP and NBR1 function in two distinct but complementary anti-proteotoxic pathways in plant stress responses.