Abstract
The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.
Highlights
Most of the proteins synthesized in the endoplasmic reticulum (ER) are glycoproteins that contain the oligosaccharide Glc3Man9GlcNAc2
Several nucleotide sugar transporters (NSTs) have been described in eukaryotes [10], most of them have been related to protein glycosylation and polysaccharide biosynthesis in the Golgi apparatus and no NST involved in supplying the substrate for UDP-glucose glycoprotein glucosyltransferase (UGGT) has yet been described
AtUTr1 Responds to the unfolded protein response (UPR)—We analyzed the expression of the AtUTr1 gene in 6-day-old plants treated with DTT, a treatment that is known to trigger the UPR given its interference in the formation of disulfide bonds [31]
Summary
Most of the proteins synthesized in the ER are glycoproteins that contain the oligosaccharide Glc3Man9GlcNAc2. Toward this end we characterized an Arabidopsis insertional mutant in the AtUTr1 gene that shows less incorporation of UDP-glucose into ER vesicles and observed that the expression level of the chaperones BiP and calnexin are constitutively up-regulated, while the expression of other genes is not. 50 g of protein corresponding to the ER vesicles from the wild type and atutr1 plants were incubated with 1 M UDP-[3H]glucose (0.1 Ci) in a medium containing 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, and 1 mM MgCl2 (STM buffer) for 3 min.
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