White dotted squares show the zoom-in area. that is present particularly in plants and beyond. homologous E3 ligase genes in (and 0.05) with total expression of at least 200 fragments per exon kilobase per million reads mapped (FKPM) (Fig. 1and and and Dataset S1). To test whether these RING E3 ligases are involved in proteotoxic stress responses, we performed serial genetic and molecular analyses. First, to perform gain-of-function analyses, the three best candidates ((overexpression lines (overexpression lines. For separation of soluble pUb proteins from insoluble pUb-protein fractions, we performed the fractionation assay with 1% Triton X-100 supplemented buffer, which was used in a previous study (36). As shown in Fig. 1is a positive regulator of PQC. Accordingly, we named the identified RING-type E3 ligase as transcripts were immediately increased by the AZC treatment, showing that is a fast-responsive gene for proteotoxic stress. and (Fig. 1and deficiency or MPSR1 activity suppression resulted in a defective PQC pathway and compromised plant survival. We treated the seedlings of MPSR1-deficient lines with AZC (100 M) for 2 wk. Then, the levels of soluble and insoluble pUb proteins were determined with -Ub antibody. We found that insoluble Rabbit Polyclonal to MMP-7 pUb proteins remarkably accumulated in the KD, DN, and lines compared with the wild-type and lines. GSK2239633A However, the soluble pUb proteins were notably changed neither by MPSR deficiency nor by MPSR1 excessivity (Fig. 1 and (lines showed increased levels of free ubiquitins (20% 90%) whereas lines showed reduced free ubiquitins under the stress condition (45%) (Fig. 1and lines ( 10%) (Fig. 1seedlings were hypersensitive to AZC treatment and their growth were severely inhibited (Fig. 1and and to remove damaged proteins before forming cytotoxic aggregates that threaten survival. Open in a separate window Fig. 1. MPSR1 is essential for plant survival under proteotoxic stress. (transcript under AZC treatment (5 mM). = 3). (lines under stress or nonstress condition. (lines under proteotoxic stress condition or nonstress condition. Protein samples were resolved by 15% SDS/PAGE. Blue arrowheads indicate free ubiquitins. Red brackets indicate ubiquitinated proteins. (lines upon AZC treatment (50, 100 M). MPSR1 Functions as a PQC E3 Ligase Independently of Cytoplasmic Chaperones. The covalent modification of misfolded proteins by ubiquitin for proteasomal degradation is the major function of PQC E3 ligases. To test whether MPSR1 has E3 ligase activity, we performed an in vitro self-ubiquitination assay, which is widely used to monitor E3 ligase activity, and we clearly observed MPSR1 self-ubiquitination (Fig. 2lines (Fig. 1and seedlings, treated with the indicated chemicals. flag-MPSR1 and associated proteins were precipitated with -flag resin. Coprecipitated catalase and flag-MPSR1 were determined with an -catalase antibody and an -flag antibody, respectively (and seedlings were used for an immunohistochemistry assay using -flag for MPSR1 and -CAT for CAT. After the AZC treatment, green GSK2239633A fluorescence from MPSR1 and red fluorescence from CAT were colocalized as speckles near the nucleus. White dotted squares show the zoom-in area. White dotted circles indicate the nucleus. The nucleus was detected with DAPI staining. The first step in all PQC pathways is the sensing of misfolded proteins, which is followed by damage assessment and a fate decision. In many cases, chaperones and cochaperones serve to recognize damaged proteins and to facilitate refolding, or if refolding does not occur, to recruit E3 ligases and the 26S proteasome for degradation (4). For GSK2239633A instance, CHIP E3 ligase forms an E3 ligaseCchaperone complex with HSP90 and HSP70 to selectively eliminate misfolded proteins in animals and plants (29, 30). Therefore, we examined whether MPSR1 is able to recognize misfolded proteins independently or dependently of chaperones. First, using yeast-two hybrid assay, we investigated whether MPSR1 forms an E3 ligaseCchaperone complex with cytoplasmic chaperones in plants. Interestingly, MPSR1 was unable to associate with five typical cytoplasmic chaperones in plants: HSP101, HSP90.1, HSP70.1, HSP40, and HSP17.4 (Fig. 2transcripts was immediately elevated ninefold, while the expression of was somewhat decreased (lines, showing that MPSR1 is able to remove arsenite-bound misfolded proteins or to prevent arsenite-induced protein aggregations (seedlings were treated under continuous strong arsenite stress for more than a week, wild-type seedlings failed to survive, while seedlings maintained their growth, showing that MPSR1 alleviates arsenite stress leading to plant viability (and (overexpressing flag-MPSR1) seedlings were treated with MG132 to accumulate pUb proteins, or with MG132/AZC to increase misfolded pUb proteins. Using an anti-flag antibody, we immunoprecipitated flag-MPSR1 (Fig. 2seedling samples (seedlings. Ubiquitinated MPSR1 proteins in the precipitates were distinctively observed as smeared bands when the activity of the.