title: Folding, quality control, and secretion of pancreatic ribonuclease in live cells.
abstract: Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t(½) < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t(½) = 16 min) to the extracellular space (t(½) = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn(113), a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.
authors.affiliation: Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Roger
authors.initials: R
authors.lastname: Geiger
authors.forename: Matthias
authors.initials: M
authors.lastname: Gautschi
authors.forename: Friederike
authors.initials: F
authors.lastname: Thor
authors.forename: Arnold
authors.initials: A
authors.lastname: Hayer
authors.forename: Ari
authors.initials: A
authors.lastname: Helenius
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D020799:Pancrelipase / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology; D017510:Protein Folding; D055503:Protein Multimerization / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*
chemical_list: D020799:Pancrelipase; D012259:Ribonuclease, Pancreatic