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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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Domain-swapped dimer. A, crystal structure of the major RNase A dimer. The N and C termini are labeled. The C-terminal β-strands of the green and blue subunits are swapped. The magnification shows the two Asn113 that form three hydrogen bonds with each other. The figure was prepared with PyMOL. B, CHO cells expressing wild-type bovine RNase, N113S bovine (bov.) RNase, or human RNase were pulsed for 4 min (0.2 mCi/ml [35S]Met/Cys) and chased for 60 min. After IP of RNase from cell lysates or medium, samples were subjected to reducing SDS-PAGE, followed by autoradiography. C, quantification of three independent experiments of B. Bars represent the means ± S.E. D, CHO cells expressing the indicated RNases were fixed and immunostained with HA and calnexin antibodies. Scale bar represents 10 μm. E, semiautomated detection of cellular outlines (green) and Golgi (yellow) with CellProfiler. F, ratio of fluorescence intensity (FI) measured from Golgi areas and cellular outlines. Bars represent means ± S.E. (n > 200 cells). MannII, mannosidase II; A, RNase A; B, RNase B.
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Figure 7: Domain-swapped dimer. A, crystal structure of the major RNase A dimer. The N and C termini are labeled. The C-terminal β-strands of the green and blue subunits are swapped. The magnification shows the two Asn113 that form three hydrogen bonds with each other. The figure was prepared with PyMOL. B, CHO cells expressing wild-type bovine RNase, N113S bovine (bov.) RNase, or human RNase were pulsed for 4 min (0.2 mCi/ml [35S]Met/Cys) and chased for 60 min. After IP of RNase from cell lysates or medium, samples were subjected to reducing SDS-PAGE, followed by autoradiography. C, quantification of three independent experiments of B. Bars represent the means ± S.E. D, CHO cells expressing the indicated RNases were fixed and immunostained with HA and calnexin antibodies. Scale bar represents 10 μm. E, semiautomated detection of cellular outlines (green) and Golgi (yellow) with CellProfiler. F, ratio of fluorescence intensity (FI) measured from Golgi areas and cellular outlines. Bars represent means ± S.E. (n > 200 cells). MannII, mannosidase II; A, RNase A; B, RNase B.

Mentions: To determine what was different about the retained versus the secreted RNase, we subjected samples of labeled CHO cell lysates and medium to size exclusion chromatography. To calibrate the column, we used purified, monomeric RNase A and B, as well as domain-swapped RNase dimers and higher oligomers prepared according to a protocol in which RNase is subjected to lyophilization from acetic acid (16). The elution profiles of monomeric RNase A and B, and RNase oligomers are shown in Fig. 6A. Oligomers generated in this way are predominantly composed of dimers in which the C-terminal β-strands (residues 116–124) are swapped between otherwise fully folded, oxidized, and enzymically active molecules (Fig. 7A) (29, 30).


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)

Domain-swapped dimer. A, crystal structure of the major RNase A dimer. The N and C termini are labeled. The C-terminal β-strands of the green and blue subunits are swapped. The magnification shows the two Asn113 that form three hydrogen bonds with each other. The figure was prepared with PyMOL. B, CHO cells expressing wild-type bovine RNase, N113S bovine (bov.) RNase, or human RNase were pulsed for 4 min (0.2 mCi/ml [35S]Met/Cys) and chased for 60 min. After IP of RNase from cell lysates or medium, samples were subjected to reducing SDS-PAGE, followed by autoradiography. C, quantification of three independent experiments of B. Bars represent the means ± S.E. D, CHO cells expressing the indicated RNases were fixed and immunostained with HA and calnexin antibodies. Scale bar represents 10 μm. E, semiautomated detection of cellular outlines (green) and Golgi (yellow) with CellProfiler. F, ratio of fluorescence intensity (FI) measured from Golgi areas and cellular outlines. Bars represent means ± S.E. (n > 200 cells). MannII, mannosidase II; 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 7: Domain-swapped dimer. A, crystal structure of the major RNase A dimer. The N and C termini are labeled. The C-terminal β-strands of the green and blue subunits are swapped. The magnification shows the two Asn113 that form three hydrogen bonds with each other. The figure was prepared with PyMOL. B, CHO cells expressing wild-type bovine RNase, N113S bovine (bov.) RNase, or human RNase were pulsed for 4 min (0.2 mCi/ml [35S]Met/Cys) and chased for 60 min. After IP of RNase from cell lysates or medium, samples were subjected to reducing SDS-PAGE, followed by autoradiography. C, quantification of three independent experiments of B. Bars represent the means ± S.E. D, CHO cells expressing the indicated RNases were fixed and immunostained with HA and calnexin antibodies. Scale bar represents 10 μm. E, semiautomated detection of cellular outlines (green) and Golgi (yellow) with CellProfiler. F, ratio of fluorescence intensity (FI) measured from Golgi areas and cellular outlines. Bars represent means ± S.E. (n > 200 cells). MannII, mannosidase II; A, RNase A; B, RNase B.
Mentions: To determine what was different about the retained versus the secreted RNase, we subjected samples of labeled CHO cell lysates and medium to size exclusion chromatography. To calibrate the column, we used purified, monomeric RNase A and B, as well as domain-swapped RNase dimers and higher oligomers prepared according to a protocol in which RNase is subjected to lyophilization from acetic acid (16). The elution profiles of monomeric RNase A and B, and RNase oligomers are shown in Fig. 6A. Oligomers generated in this way are predominantly composed of dimers in which the C-terminal β-strands (residues 116–124) are swapped between otherwise fully folded, oxidized, and enzymically active molecules (Fig. 7A) (29, 30).

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