Accelerated secretion of mutant β-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution

Transformed yeasts producing a mutant form of bovine β-lactoglobulin (β-LG), W19Y, in which Trp¹⁹ was replaced with Tyr, were shown to secrete 6 times more than those producing wild type β-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type β-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type β-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type β-LG. Our previous study showed that the ΔG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type β-LG. Furthermore, immunoblot analysis of intracellular β-LG under non-reducing conditions indicated that W19Y as well as wild type β-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant β-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type β-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type β-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure. Copyright (C) 1999 Elsevier Science B.V.