A fraction of each secreted protein is retained and degraded by the endoplasmic reticulum (ER) quality control apparatus that restricts export to correctly folded proteins. The intrinsic biophysical attributes that determine efficiency of escape from this proofreading process have been examined by expressing mutants of bovine pancreatic trypsin inhibitor (BPTI) in yeast. Secretion efficiency is strongly correlated with thermodynamic stability for a series of six point mutations of BPTI. No correlation of secretion efficiency with either oxidative folding or refolding rates in vitro is found; both the rapidly folded Y35L BPTI mutant and the slowly unfolded G36D BPTI mutant exhibit low secretion efficiency. Elimination of cysteines 14 and 38 by mutagenesis does not increase secretion efficiency, indicating that intramolecular thiol/disulfide rearrangements are not primarily responsible for retention and degradation of destabilized BPTI variants. Mutant yeast strains with diminished ER-associated degradation do not secrete BPTI more efficiently, indicating that retention and degradation are separable processes. These data support a model for ER quality control, wherein protein folding is functionally reversible and the relative rates of folding, unfolding, vesicular export, and retention determine secretion efficiency.

Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion. Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.

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