COMPETITION BETWEEN FOLDING AND GLYCOSYLATION IN THE ENDOPLASMIC-RETICULUM

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system , the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at p ositions buried in the folded protein structure. The level of glycosyl ation of such new acceptor sites was analysed by pulse-labelling under two sets of conditions that are known to reduce the rate of folding: (i) addition of dithiothreitol to the growth medium and (ii) introduct ion of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, a ll the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting c ategory of mutants, the level of glycosylation vas dependent on the co nditions for folding. This shows that folding and glycosylation reacti ons can compete in vivo and that glycosylation does not necessarily pr ecede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.