The quality control of glycoprotein folding in the endoplasmic reticulum (ER) involves the interplay of a glucosyltransferase (GT) that only glycosylates not properly folded protein conformers, glucosidase lI (GII) that removes residues added by GT and two ER resident lectins (calnexin, CNX and calreticulin, CRT) that specifically recognize monoglucosylated glycoproteins. This mechanism prevents exit of not properly folded glycoproteins to the Golgi and enhances glycoprotein folding efficiency. We have recently obtained evidence indicating that GT recognizes hydrophobic amino acid patches exposed in molten globule-like conformations displayed at the last glycoprotein folding stages. The purpose of Specific Aim I is to study, at the molecular level, glycoprotein interaction with GT-CNX/CRT and other ER chaperones, from the time the polypeptide moiety emerges into the ER lumen until an oligomer complex is formed. As UDP-Glc is GT substrate donor, the glucosylating reaction produces UDP as one of its reaction products. Nucleoside diphosphates generated by glycosyltransferases in the secretory pathway must be converted into monophosphates to relieve inhibition of the transferring enzymes and to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to exit of nucleoside monophosphate. We have recently obtained evidence suggesting that in the yeast Schizosaccharomyces pombe GT-generated UDP might be hydrolyzed by nucleoside diphosphatases occurring not in the ER but in the Golgi apparatus.
Specific Aim II deals with the possibility that vesicular transport between the ER and cis Golgi cisternae might carry not only the macromolecular components of the quality control mechanism (GT, GII, CNX and CRT have ER retrieval sequences at their C-termini) but also UDP in the anterograd movement to be hydrolyzed by Golgi GDPases/UDPases and LIMP in the retrograd movement. Finally, Specific Aim III deals on the mechanism, intimately intertwined with that of quality control, by which cells distinguish between folding intermediates that are in the process of productive folding and irremediably misfolded glycoproteins that have to be diverted to proteasomal degradation.
