TY - JOUR

T1 - Competition between folding and glycosylation in the endoplasmic reticulum

AU - Holst, B

AU - Bruun, A W

AU - Kielland-Brandt, Morten

AU - Winther, Jakob R.

PY - 1996

Y1 - 1996

N2 - 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 positions buried in the folded protein structure. The level of glycosylation 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) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.

AB - 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 positions buried in the folded protein structure. The level of glycosylation 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) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Binding Sites

KW - Binding, Competitive

KW - Carboxypeptidases

KW - Cathepsin A

KW - DNA, Fungal

KW - Endoplasmic Reticulum

KW - Glycosylation

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Folding

KW - Saccharomyces cerevisiae

M3 - Journal article

C2 - 8670857

VL - 15

SP - 3538

EP - 3546

JO - E M B O Journal

JF - E M B O Journal

SN - 0261-4189

IS - 14

ER -