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Folding, quality control, and secretion of pancreatic ribonuclease in live cells.

Geiger R, Gautschi M, Thor F, Hayer A, Helenius A - J. Biol. Chem. (2010)

Bottom Line: 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%.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland.

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.

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Bovine and human RNase are secreted rapidly but with different efficiency. A, CHO cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) for 2 min and chased for the indicated times. After IP of RNase from cell lysates or media with RNase antibody, samples were subjected to Endo H digestion followed by separation on reducing SDS-PAGE and autoradiography. B, the percentage of Endo H-resistant RNase B was plotted versus chase time and was fitted with a Boltzmann sigmoidal curve. A half-time of 16 min was calculated. Error bars represent the S.E. C, secreted RNase after a 60-min chase was used as a control. D, one representative example of three independent pulse-chase experiments of human and bovine RNase in CHO cells. After IP of RNase from cell lysate or medium, samples were subjected to reducing SDS-PAGE followed by autoradiography (quantification by ImageJ). E, the fraction of secreted RNase of total RNase (cells and medium) was plotted versus chase time. Data points represent means ± S.E. and were fitted with a Boltzmann sigmoidal curve. A, RNase A; B, RNase B.
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Figure 4: Bovine and human RNase are secreted rapidly but with different efficiency. A, CHO cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) for 2 min and chased for the indicated times. After IP of RNase from cell lysates or media with RNase antibody, samples were subjected to Endo H digestion followed by separation on reducing SDS-PAGE and autoradiography. B, the percentage of Endo H-resistant RNase B was plotted versus chase time and was fitted with a Boltzmann sigmoidal curve. A half-time of 16 min was calculated. Error bars represent the S.E. C, secreted RNase after a 60-min chase was used as a control. D, one representative example of three independent pulse-chase experiments of human and bovine RNase in CHO cells. After IP of RNase from cell lysate or medium, samples were subjected to reducing SDS-PAGE followed by autoradiography (quantification by ImageJ). E, the fraction of secreted RNase of total RNase (cells and medium) was plotted versus chase time. Data points represent means ± S.E. and were fitted with a Boltzmann sigmoidal curve. A, RNase A; B, RNase B.

Mentions: To determine the kinetics and efficiency by which RNase reached the Golgi complex, we took advantage of the observation that the secreted RNase B was Endo H-resistant (Fig. 4C), i.e. the N-linked glycan was converted to the complex type. Conversion occurs in the medial Golgi (25). A pulse-chase experiment showed that the first Endo H-resistant RNase B molecules appeared after 10 min chase (Fig. 4A). The half-time of conversion was 16 min, and the maximum level was reached after 30 min (Fig. 4B).


Folding, quality control, and secretion of pancreatic ribonuclease in live cells.

Geiger R, Gautschi M, Thor F, Hayer A, Helenius A - J. Biol. Chem. (2010)

Bovine and human RNase are secreted rapidly but with different efficiency. A, CHO cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) for 2 min and chased for the indicated times. After IP of RNase from cell lysates or media with RNase antibody, samples were subjected to Endo H digestion followed by separation on reducing SDS-PAGE and autoradiography. B, the percentage of Endo H-resistant RNase B was plotted versus chase time and was fitted with a Boltzmann sigmoidal curve. A half-time of 16 min was calculated. Error bars represent the S.E. C, secreted RNase after a 60-min chase was used as a control. D, one representative example of three independent pulse-chase experiments of human and bovine RNase in CHO cells. After IP of RNase from cell lysate or medium, samples were subjected to reducing SDS-PAGE followed by autoradiography (quantification by ImageJ). E, the fraction of secreted RNase of total RNase (cells and medium) was plotted versus chase time. Data points represent means ± S.E. and were fitted with a Boltzmann sigmoidal curve. A, RNase A; B, RNase B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037694&req=5

Figure 4: Bovine and human RNase are secreted rapidly but with different efficiency. A, CHO cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) for 2 min and chased for the indicated times. After IP of RNase from cell lysates or media with RNase antibody, samples were subjected to Endo H digestion followed by separation on reducing SDS-PAGE and autoradiography. B, the percentage of Endo H-resistant RNase B was plotted versus chase time and was fitted with a Boltzmann sigmoidal curve. A half-time of 16 min was calculated. Error bars represent the S.E. C, secreted RNase after a 60-min chase was used as a control. D, one representative example of three independent pulse-chase experiments of human and bovine RNase in CHO cells. After IP of RNase from cell lysate or medium, samples were subjected to reducing SDS-PAGE followed by autoradiography (quantification by ImageJ). E, the fraction of secreted RNase of total RNase (cells and medium) was plotted versus chase time. Data points represent means ± S.E. and were fitted with a Boltzmann sigmoidal curve. A, RNase A; B, RNase B.
Mentions: To determine the kinetics and efficiency by which RNase reached the Golgi complex, we took advantage of the observation that the secreted RNase B was Endo H-resistant (Fig. 4C), i.e. the N-linked glycan was converted to the complex type. Conversion occurs in the medial Golgi (25). A pulse-chase experiment showed that the first Endo H-resistant RNase B molecules appeared after 10 min chase (Fig. 4A). The half-time of conversion was 16 min, and the maximum level was reached after 30 min (Fig. 4B).

Bottom Line: 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%.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland.

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.

Show MeSH
Related in: MedlinePlus