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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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Folding of bovine RNase occurs rapidly in the ER of CHO cells. Cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) with or without chase (times are indicated). Folding was assayed by trypsin resistance of RNase. After IP of RNase molecules with RNase antibody from the media (A) or cell lysates (B and C), samples were subjected to trypsin digestion for 5 min at 20 °C followed by reducing SDS-PAGE and autoradiography. D and E, pulse-chase (0.5 mCi/ml [35S]Met/Cys) of bovine RNase in CHO cells to determine folding after DTT washout and readdition. Cells were labeled in the presence of DTT (4 min) and subsequently chased in medium lacking DTT for the time indicated (chase without DTT), followed by a chase for 60 min in medium supplemented with DTT. After IP of secreted RNase with RNase antibody from the medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. F, quantification of secreted RNase molecules. The amount of secreted RNase after 15 min of folding time in the absence of DTT was set to 1. The other data points were normalized to it and fitted with a Boltzmann sigmoidal curve. A half-time of 2.1 min was calculated. A, RNase A; B, RNase B.
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Figure 2: Folding of bovine RNase occurs rapidly in the ER of CHO cells. Cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) with or without chase (times are indicated). Folding was assayed by trypsin resistance of RNase. After IP of RNase molecules with RNase antibody from the media (A) or cell lysates (B and C), samples were subjected to trypsin digestion for 5 min at 20 °C followed by reducing SDS-PAGE and autoradiography. D and E, pulse-chase (0.5 mCi/ml [35S]Met/Cys) of bovine RNase in CHO cells to determine folding after DTT washout and readdition. Cells were labeled in the presence of DTT (4 min) and subsequently chased in medium lacking DTT for the time indicated (chase without DTT), followed by a chase for 60 min in medium supplemented with DTT. After IP of secreted RNase with RNase antibody from the medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. F, quantification of secreted RNase molecules. The amount of secreted RNase after 15 min of folding time in the absence of DTT was set to 1. The other data points were normalized to it and fitted with a Boltzmann sigmoidal curve. A half-time of 2.1 min was calculated. A, RNase A; B, RNase B.

Mentions: To determine the rate of folding, we took advantage of the conversion of RNase from a protease-sensitive to a protease-resistant form (11, 20). The RNase secreted into the medium was fully resistant to trypsin as shown in (Fig. 2A). Less than 10% of the labeled protein was resistant at the end of a 1-min pulse (Fig. 2B). However, after a 4-min pulse 80%, and after a further 4 min of chase, 95% of the labeled protein was resistant. This indicated that bovine RNase folded to a trypsin-resistant form with a half time of less than 3 min after completed synthesis.


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)

Folding of bovine RNase occurs rapidly in the ER of CHO cells. Cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) with or without chase (times are indicated). Folding was assayed by trypsin resistance of RNase. After IP of RNase molecules with RNase antibody from the media (A) or cell lysates (B and C), samples were subjected to trypsin digestion for 5 min at 20 °C followed by reducing SDS-PAGE and autoradiography. D and E, pulse-chase (0.5 mCi/ml [35S]Met/Cys) of bovine RNase in CHO cells to determine folding after DTT washout and readdition. Cells were labeled in the presence of DTT (4 min) and subsequently chased in medium lacking DTT for the time indicated (chase without DTT), followed by a chase for 60 min in medium supplemented with DTT. After IP of secreted RNase with RNase antibody from the medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. F, quantification of secreted RNase molecules. The amount of secreted RNase after 15 min of folding time in the absence of DTT was set to 1. The other data points were normalized to it and fitted with a Boltzmann sigmoidal curve. A half-time of 2.1 min was calculated. 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 2: Folding of bovine RNase occurs rapidly in the ER of CHO cells. Cells expressing bovine RNase were pulse-labeled (0.5 mCi/ml [35S]Met/Cys) with or without chase (times are indicated). Folding was assayed by trypsin resistance of RNase. After IP of RNase molecules with RNase antibody from the media (A) or cell lysates (B and C), samples were subjected to trypsin digestion for 5 min at 20 °C followed by reducing SDS-PAGE and autoradiography. D and E, pulse-chase (0.5 mCi/ml [35S]Met/Cys) of bovine RNase in CHO cells to determine folding after DTT washout and readdition. Cells were labeled in the presence of DTT (4 min) and subsequently chased in medium lacking DTT for the time indicated (chase without DTT), followed by a chase for 60 min in medium supplemented with DTT. After IP of secreted RNase with RNase antibody from the medium, samples were subjected to reducing SDS-PAGE followed by autoradiography. F, quantification of secreted RNase molecules. The amount of secreted RNase after 15 min of folding time in the absence of DTT was set to 1. The other data points were normalized to it and fitted with a Boltzmann sigmoidal curve. A half-time of 2.1 min was calculated. A, RNase A; B, RNase B.
Mentions: To determine the rate of folding, we took advantage of the conversion of RNase from a protease-sensitive to a protease-resistant form (11, 20). The RNase secreted into the medium was fully resistant to trypsin as shown in (Fig. 2A). Less than 10% of the labeled protein was resistant at the end of a 1-min pulse (Fig. 2B). However, after a 4-min pulse 80%, and after a further 4 min of chase, 95% of the labeled protein was resistant. This indicated that bovine RNase folded to a trypsin-resistant form with a half time of less than 3 min after completed synthesis.

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