Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglu cosylated oligosaccharides. Binding and release are regulated by two enzyme s, glucosidase II and UDP-Glc: glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-lin ked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins, To investigate how nonnative protein confor mations are recognized and directed to this unique chaperone system, we ana lyzed the interaction of GT with a series of model substrates with well def ined conformations derived from RNaseB. We found that conformations with sl ight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When th is form was converted back to a nativelike state, concomitant loss of recog nition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish bet ween different nonnative conformations with a distinct preference for parti ally structured conformers, The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.