PDI improves secretion of redox-inactive beta-glucosidase.
Although manipulation of the endoplasmic reticulum (ER) folding environment in the yeast Saccharomyces cerevisiae has been shown to increase the secretory productivity of recombinant proteins, the cellular interactions and processes of native enzymes and chaperones such as protein disulfide isomerase (PDI) are still unclear. Previously, we reported that overexpression of the ER chaperone PDI enabled up to a 3-fold increase in secretion levels of the Pyrococcus furiosus beta-glucosidase in the yeast S. cerevisiae. This result was surprising since beta-glucosidase contains only one cysteine per monomer and no disulfide bonds. Two possible mechanisms were proposed: PDI either forms a transient disulfide bond with the lone cysteine residue of the nascent beta-glucosidase during the folding and assembly process or acts as a chaperone to aid in proper folding. To discern between the two mechanisms, the single cysteine residue was mutated to serine, and the secretion of the two protein variants was determined. The serine mutant still showed increased secretion in vivo when PDI levels were elevated. When the folding bottleneck is removed by increasing expression temperatures to 37 degrees C rather than 30 degrees C, PDI no longer has an improvement on secretion. These results suggest that, unexpectedly, PDI acts in a chaperone-like capacity or possibly cooperates with the cell's folding or degradation mechanisms regardless of whether the protein is redox-active.
Powers
Sara Lawrence
SL
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, Delaware 19716, USA.
Robinson
Anne Skaja
AS
D024861:Genetic Enhancement / Q000379:methods; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D020027:Pyrococcus furiosus / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D001617:beta-Glucosidase; D019704:Protein Disulfide-Isomerases