Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDI A was previously isolated from Aspergillus niger, and rye report its functi onal characterization here. Functional analysis of PDIA showed that it cata lyzes the refolding of denatured and reduced RNase A. pdiA also complemente d PDI function in a Saccharomyces cerevisiae Delta pdi1 mutant in a yeast-b ased killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised b y the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the pri mary stress response. An increased level of pdiA transcripts was also obser ved in two A. niger strains overproducing a heterologous protein, hen egg w hite lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpr ession of PDIA did not increase secreted yields of HEWL in A. niger, sugges ting that PDIA itself is not limiting for secretion of this protein. Downre gulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA ac tivity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.