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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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Monomeric RNase is secreted, whereas dimers are retained. A, gel filtration of RNase A monomers and domain-swapped dimers and trimers of RNase A and B. A mixture of monomers, dimers, and higher oligomers was prepared from a partially purified RNase A and B fraction by lyophilization according to the protocol by Crestfield et al. (16) and subjected to Superdex 75 gel filtration (red profile). Untreated RNase A momomers were also analyzed on the same column (black elution profile). Untreated RNase A peaked in fraction (F) 27. RNase dimers peaked in fraction 23, monomeric RNase B peaked in fraction 25, monomeric RNase A peaked in fraction 27. B, CHO cells expressing bovine RNase were pulsed for 15 min (0.5 mCi/ml [35S]Met/Cys). After labeling, the cells were lysed. A sample of the lysate (T) was removed, and the rest was applied to a Superdex 75 gel filtration column. The lysate sample (T) and the eluted fractions were subjected to IP using polyclonal RNase antibody followed by reducing SDS-PAGE and autoradiography. The bands corresponding to dimeric RNases (arrowheads) were resistant to SDS and DTT. C and D, cells were pulsed for 15 min and chased for 60 min. Both media and cell lysates were fractionated on a Superdex 75 gel filtration column. Note that only the monomeric RNase was secreted. To visualize the low levels of cell-associated bovine RNase, the gels were exposed longer than in B. E, same as in C, but fractions were subjected to IP using polyclonal RNase antibody followed by SDS denaturation and additional IP using HA.11 antibody against the HA-tagged RNase. F, pancreatic tissue was homogenized in a buffer containing 2% CHAPS and fractionated on a Superdex 75 column. Fractions were subjected to IP using RNase antibodies and immunoblotted with RNase antibodies. Only few dimers were resistant to SDS and DTT (arrowhead). G, pancreatic juice was fractionated and analyzed like the tissue in F. A, RNase A; B, RNase B.
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Figure 6: Monomeric RNase is secreted, whereas dimers are retained. A, gel filtration of RNase A monomers and domain-swapped dimers and trimers of RNase A and B. A mixture of monomers, dimers, and higher oligomers was prepared from a partially purified RNase A and B fraction by lyophilization according to the protocol by Crestfield et al. (16) and subjected to Superdex 75 gel filtration (red profile). Untreated RNase A momomers were also analyzed on the same column (black elution profile). Untreated RNase A peaked in fraction (F) 27. RNase dimers peaked in fraction 23, monomeric RNase B peaked in fraction 25, monomeric RNase A peaked in fraction 27. B, CHO cells expressing bovine RNase were pulsed for 15 min (0.5 mCi/ml [35S]Met/Cys). After labeling, the cells were lysed. A sample of the lysate (T) was removed, and the rest was applied to a Superdex 75 gel filtration column. The lysate sample (T) and the eluted fractions were subjected to IP using polyclonal RNase antibody followed by reducing SDS-PAGE and autoradiography. The bands corresponding to dimeric RNases (arrowheads) were resistant to SDS and DTT. C and D, cells were pulsed for 15 min and chased for 60 min. Both media and cell lysates were fractionated on a Superdex 75 gel filtration column. Note that only the monomeric RNase was secreted. To visualize the low levels of cell-associated bovine RNase, the gels were exposed longer than in B. E, same as in C, but fractions were subjected to IP using polyclonal RNase antibody followed by SDS denaturation and additional IP using HA.11 antibody against the HA-tagged RNase. F, pancreatic tissue was homogenized in a buffer containing 2% CHAPS and fractionated on a Superdex 75 column. Fractions were subjected to IP using RNase antibodies and immunoblotted with RNase antibodies. Only few dimers were resistant to SDS and DTT (arrowhead). G, pancreatic juice was fractionated and analyzed like the tissue in F. 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)

Monomeric RNase is secreted, whereas dimers are retained. A, gel filtration of RNase A monomers and domain-swapped dimers and trimers of RNase A and B. A mixture of monomers, dimers, and higher oligomers was prepared from a partially purified RNase A and B fraction by lyophilization according to the protocol by Crestfield et al. (16) and subjected to Superdex 75 gel filtration (red profile). Untreated RNase A momomers were also analyzed on the same column (black elution profile). Untreated RNase A peaked in fraction (F) 27. RNase dimers peaked in fraction 23, monomeric RNase B peaked in fraction 25, monomeric RNase A peaked in fraction 27. B, CHO cells expressing bovine RNase were pulsed for 15 min (0.5 mCi/ml [35S]Met/Cys). After labeling, the cells were lysed. A sample of the lysate (T) was removed, and the rest was applied to a Superdex 75 gel filtration column. The lysate sample (T) and the eluted fractions were subjected to IP using polyclonal RNase antibody followed by reducing SDS-PAGE and autoradiography. The bands corresponding to dimeric RNases (arrowheads) were resistant to SDS and DTT. C and D, cells were pulsed for 15 min and chased for 60 min. Both media and cell lysates were fractionated on a Superdex 75 gel filtration column. Note that only the monomeric RNase was secreted. To visualize the low levels of cell-associated bovine RNase, the gels were exposed longer than in B. E, same as in C, but fractions were subjected to IP using polyclonal RNase antibody followed by SDS denaturation and additional IP using HA.11 antibody against the HA-tagged RNase. F, pancreatic tissue was homogenized in a buffer containing 2% CHAPS and fractionated on a Superdex 75 column. Fractions were subjected to IP using RNase antibodies and immunoblotted with RNase antibodies. Only few dimers were resistant to SDS and DTT (arrowhead). G, pancreatic juice was fractionated and analyzed like the tissue in F. A, RNase A; B, RNase B.
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Figure 6: Monomeric RNase is secreted, whereas dimers are retained. A, gel filtration of RNase A monomers and domain-swapped dimers and trimers of RNase A and B. A mixture of monomers, dimers, and higher oligomers was prepared from a partially purified RNase A and B fraction by lyophilization according to the protocol by Crestfield et al. (16) and subjected to Superdex 75 gel filtration (red profile). Untreated RNase A momomers were also analyzed on the same column (black elution profile). Untreated RNase A peaked in fraction (F) 27. RNase dimers peaked in fraction 23, monomeric RNase B peaked in fraction 25, monomeric RNase A peaked in fraction 27. B, CHO cells expressing bovine RNase were pulsed for 15 min (0.5 mCi/ml [35S]Met/Cys). After labeling, the cells were lysed. A sample of the lysate (T) was removed, and the rest was applied to a Superdex 75 gel filtration column. The lysate sample (T) and the eluted fractions were subjected to IP using polyclonal RNase antibody followed by reducing SDS-PAGE and autoradiography. The bands corresponding to dimeric RNases (arrowheads) were resistant to SDS and DTT. C and D, cells were pulsed for 15 min and chased for 60 min. Both media and cell lysates were fractionated on a Superdex 75 gel filtration column. Note that only the monomeric RNase was secreted. To visualize the low levels of cell-associated bovine RNase, the gels were exposed longer than in B. E, same as in C, but fractions were subjected to IP using polyclonal RNase antibody followed by SDS denaturation and additional IP using HA.11 antibody against the HA-tagged RNase. F, pancreatic tissue was homogenized in a buffer containing 2% CHAPS and fractionated on a Superdex 75 column. Fractions were subjected to IP using RNase antibodies and immunoblotted with RNase antibodies. Only few dimers were resistant to SDS and DTT (arrowhead). G, pancreatic juice was fractionated and analyzed like the tissue in F. 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