Fig 2: Nuclear-localized CTR1 stabilizes EIN3 via a non-catalytic function.a CTR1 interacts with EBF1 and EBF2, but not EIN3 in yeast-two-hybrid assays. b BiFC assay for full-length wild-type CTR1 and nuclear ethylene signaling proteins in N. benthamiana in the presence of ACC. COP1-interacting protein 8 (CIP8) and bZIP-RFP were used as a negative control and a nuclear subcellular marker, respectively. c Co-immunoprecipitation analysis of CTR1 and EBF2 in N. benthamiana. d Three-day-old dark-grown seedlings were treated with the indicated concentrations of ACC for 2 h, and total protein extracts were used for immunoblotting with anti-EIN3, GFP, and Hsc70 antibodies. e, f ctr1-8 (e) and seedlings expressing GFP-CTR1-8-SV40 NLS in the ctr1-8 mutant (f) expressed higher basal levels of EIN3 than wild-type seedlings. The experiments were repeated at least three times with similar results. g 35S:GFP-?NT-gCTR1ctr1-1 expressed a significantly higher level of EIN3 protein than the wild type after removal of ACC. Seedlings pretreated with 200 µM ACC for 2 h and total protein extracts were harvested at the indicated times after ACC removal. Rel. quantities represent the ratio of the intensity of the EIN3 band to Hsc70 band signals, and these values are expressed relative to the intensity of EIN3/Hsc70 in the wild type with that in time 0 samples, which was set to 1. h ACC dose-response curves for the hypocotyl length of 3-d-old dark-grown wild-type and 35S:GFP-?NT-CTR1ctr1-1 seedlings. Control treatments included no ACC. Error bars, SE (n = 24 seedlings for each ACC concentrations). Two-tailed Student’s t test. i Seedlings expressing GFP-?NT-gCTR1ctr1-1 from its native promoter in ctr1-2 mutant background showed an enhanced ACC response compared to CTR1p:GFP- gCTR1ctr1-1/ctr1-2 seedlings in the dark. Error bars, SE (n = 46 seedlings for each ACC concentrations). Two tailed Student’s t test. j CTR1 does not phosphorylate EBF2. In vitro kinase assay for purified ?NT-CTR1 or ?NT-CTR1ctr1-1 with EIN2-CEND, EIN3, or EBF2. EIN2-CEND and EIN3 were used as a positive and negative control, respectively. The indicated proteins were incubated together in kinase reaction buffer and then separated by SDS/PAGE, and the incorporated radiolabel was detected by autoradiography. Experiments were repeated three times with the similar results.

Fig 3: Model for ethylene-induced CTR1 nuclear translocation and stimulation of the ethylene response.In the absence of ethylene, the ethylene receptors, CTR1, and EIN2 mainly localize to the ER. Receptor-activated CTR1 phosphorylates EIN2, thus leading to the proteolytic degradation of EIN2 via EIN2-TARGETING PROTEIN 1 (ETP1) and ETP2. In the presence of ethylene, the receptors, and hence CTR1, are inactivated. Inactivated CTR1 no longer phosphorylates EIN2, and the C-terminal domain of EIN2 (EIN2-CEND) is proteolytically cleaved and translocates to the nucleus, where it initiates the primary ethylene response via EIN3 activation. EIN2-CEND is also targeted to processing bodies (P-bodies) and suppresses EBF mRNA translation. After the nuclear translocation of EIN2-CEND, CTR1 is released from the receptors and relocates to the nucleus via an unknown mechanism. The nuclear-localized CTR1 directly interacts with EBF2, thus stabilizing EIN3 via an unknown mechanism and ultimately stimulating the ethylene response. The yellow square with a question mark indicates an unknown cargo protein that delivers CTR1 to the nucleus. Arrows indicate the movement of ethylene signaling components or positive influence; blunted ends indicate inhibition.