UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with nonphysiological substrates revealed that UGGT can glucosylate nonnative glycoproteins by recognizing subtle folding defects; however, its physiological function remains undefined. Here, we show that mutations in the Arabidopsis EBS1 gene suppressed the growth defects of a brassinosteroid (BR) receptor mutant, bri1-9, in an allele-specific manner by restoring its BR sensitivity. Using a map-based cloning strategy, we discovered that EBS1 encodes the Arabidopsis homolog of UGGT. We demonstrated that bri1-9 is retained in the ER through interactions with several ER chaperones and that ebs1 mutations significantly reduce the stringency of the retention-based ER quality control, allowing export of the structurally imperfect yet biochemically competent bri1-9 to the cell surface for BR perception. Thus, our discovery provides genetic support for a physiological role of UGGT in high-fidelity ER quality control.
GLYCOGEN SYNTHASE KINASE3 (GSK3) is a highly conserved serine/threonine kinase involved in a variety of developmental signaling processes. The Arabidopsis (Arabidopsis thaliana) genome encodes 10 GSK3-like kinases that are clustered into four groups. Forward genetic screens have so far uncovered eight mutants, all of which carry gain-of-function mutations in BRASSINOSTEROID-INSENSITIVE2 (BIN2), one of the three members in group II. Genetic and biochemical studies have implicated a negative regulatory role for BIN2 in brassinosteroid (BR) signaling. Here, we report the identification of eight ethyl methanesulfonate-mutagenized loss-of-function bin2 alleles and one T-DNA insertional mutation each for BIN2 and its two closest homologs, BIN2-Like1 and BIN2-Like2. Our genetic, biochemical, and physiological assays revealed that despite functional redundancy, BIN2 plays a dominant role among the three group II members in regulating BR signaling. Surprisingly, the bin2bil1bil2 triple T-DNA insertional mutant still responds to BR and accumulates a more phosphorylated form of a BIN2 substrate than the wild-type plant. Using the specific GSK3 inhibitor lithium chloride, we have provided strong circumstantial evidence for the involvement of other Arabidopsis GSK3-like kinases in BR signaling. Interestingly, lithium chloride treatment was able to suppress the gain-of-function bin2-1 mutation but had a much weaker effect on a strong BR receptor mutant, suggesting the presence of a BIN2-independent regulatory step downstream of BR receptor activation.GLYCOGEN SYNTHASE KINASE3 (GSK3) is a highly conserved Ser/Thr kinase that is implicated in a wide range of cellular and developmental processes (Woodgett, 2001). In mammals, GSK3 has only two isoforms, GSK3a and GSK3b. By contrast, many plant species contain a much larger set of GSK3-like kinases (Richard et al., 2005;Yoo et al., 2006). Arabidopsis (Arabidopsis thaliana) has 10 GSK3-like kinases, also known as AtSKs for Arabidopsis SHAGGY-like protein kinases, that can be classified into four subgroups based on phylogeny analysis (Jonak and Hirt, 2002). Genetic, transgenic, and biochemical approaches have implicated plant GSK3-like kinases in a variety of plant signaling processes, including flower development, stress/wounding responses, and hormone signaling (Jonak and Hirt, 2002). For example, transgenic Arabidopsis plants with reduced transcript levels of AtSK1-1 and AtSK1-2 contain increased numbers of floral organs and exhibit abnormal apical-basal patterning in gynoecium (Dornelas et al., 2000), whereas overexpression of mutated AtSK3-2 displayed smaller floral organs (Claisse et al., 2007). It was also known that wounding can activate an alfalfa (Medicago sativa) GSK3 kinase (Jonak et al., 2000) and that overexpression of AtSK2-2 can complement a salt stress-sensitive mutant in yeast and results in enhanced salt resistance in Arabidopsis (Piao et al., 1999(Piao et al., , 2001.The best studied Arabidopsis GSK3-like kinase is BIN2/UCU1/DWF12/AtSK2-1, which was believed to r...
Glycogen synthase kinase 3 (GSK3) is a unique serine/threonine kinase that is implicated in a variety of cellular processes and is regulated by phosphorylation or protein-protein interaction in animal cells. BIN2 is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling. Genetic studies suggested that BIN2 is inhibited in response to BR perception at the cell surface to relieve its inhibitory effects on downstream targets; however, little is known about biochemical mechanisms of its inhibition. Here, we show that BIN2 is regulated by proteasome-mediated protein degradation. Exogenous application of a BR biosynthesis inhibitor and an active BR increased and decreased the amount of BIN2 proteins, respectively. Interestingly, the gain-of-function bin2-1 mutation significantly stabilizes BIN2, making it unresponsive to BR-induced BIN2 depletion. Exogenous application of different plant growth hormones revealed that BIN2 depletion is specifically induced by BR through a functional BR receptor, while treatment of a proteasome inhibitor, MG132, not only prevented the BR-induced BIN2 depletion but also nullified the inhibitory effect of BR on the BIN2 kinase activity. Taken together, our results strongly suggest that proteasome-mediated protein degradation constitutes an important regulatory mechanism for restricting the BIN2 activity.
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