Abstract
N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.
Highlights
Most of the secretory proteins in eucaryotes are cotranslachains in the mechanism of protein folding
In the case of oligomeric proteins, these monomers associate to the functional native state (Jaenicke1,984,1987).Domain pairing and subunit association are often found to be slow processes and tocontrol overall refolding i n vitro (Teschner et al, 1987; Vaucheret et al, 1987).Experimental data on the mechanism of the i n vivo folding of nascent protein chains are scarce
Analysis of the kinetic curves implies that under these conditions unfolding of both invertase forms has gone to completion within about 15min
Summary
Most of the secretory proteins in eucaryotes are cotranslachains in the mechanism of protein folding. Glycosylation improves the solubilitoyf un- for sugar chainsare located at the surface of the folded foldedorpartiallyfoldedinvertasemoleculesand protein, as judged from the known three-dimensional struchence leads to a suppression of irreversible aggrega- ture of bovine ribonuclease A, which is free of carbohydrate tion Such a protective effect may be important (Wlodawer and Sjolin, 1983; Beintema, 1986). In the case of oligomeric proteins, these monomers associate to the functional native state (Jaenicke1,984,1987).Domain pairing and subunit association are often found to be slow processes and tocontrol overall refolding i n vitro (Teschner et al, 1987; Vaucheret et al, 1987).Experimental data on the mechanism of the i n vivo folding of nascent protein chains are scarce. In the preceding study (Schdke andSchmid, 1988),we compared the stability toward unfolding of both invertasesby heat and by denaturants and measured the rates of unfolding under denaturing conditions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
