Expression and secretion of human protein disulphide isomerase in Saccharomyces cerevisiae.

Protein disulphide isomerase (PDV), an enzyme which catalyses thio1:disulphide interchange, is a major protein of the endoplasmic reticulum (ER) of plants, animals and yeast [1,2,3]. This enzyme seems to be critical for attaining the correctly folded tertiary structure of disulphide-bonded proteins both in vivo and in vitro. PDI is ubiquitous, but its activity is highest in cells actively producing secretory proteins [4]. Most ER lumenal proteins have a C-terminal tetra-peptide which functions as an ER retention signal [5]. This tetra-peptide (HDEL in yeast and KDEL in mammalian cells, [6]) does not prevent ER lumenal proteins from acquiring post-translational modifications characteristic of early compartements of the Golgi apparatus [6,7], which suggests that they can be retrieved to the ER. A receptor for ER proteins, that cycles between the ER and the Golgi apparatus, has been found in yeast [8]. The human PDI gene codes for a protein that acts simultaneously as PDI and the P-subunit of prolyl-4hydroxylase (P4H, [9]). Recent reports suggest that the main function of the P4H P-subunit is to retain the protein in the ER lumen by means of its KDEL signal [10,11]. A variety of other functions have been described for PDI (see [12], for a review). PDI, isolated in solution is a homodimer [5], and the human monomer is 55-60kDa [ 1 1,13,14]. The yeast enzyme is ,70kDa, with a shift to 6OkDa after endoglycosidase H treatment, indicating that the protein is N-glycosylated [15]. In this study we describe the expression of functional hPDl in the yeast S.cerevisiae and demonstrate that the tetra-peptide C-terminal retention signal can be overridden in yeast by either overexpression or by use of the mating factor a (MFa) pre-pro sequence at the N-terminus of the polypeptide. A hPDl expression cassette was integrated at the Lys2 locus of Saccharomyces cerevisiae. The expression cassette consisted of the GAL1 .I 0 promoter, the leader sequence of the MFa fused in frame with the sequence for mature hPDI, with either the native C-terminal KDEL sequence or a mutated sequence coding for HDEL signal. Yeast strains were grown in 3YEPD (4.8% glucose, 6% Oxoid yeast extract, 3Oh Bacto peptone), for 24 hours at 30"C, and then transferred to 3YGAL (3YEPD with glucose substituted by galactose) for a further 24 hours. Protein extraction was achieved by glass bead lysis, and for analysis of secreted proteins, the culture medium was precipitated by addition of TCA to a final concentration of 6%. After SDS-PAGE analysis, proteins were transferred to a nitrocellulose membrane and incubated with antibodies raised against bPDl [16], yPDl [17] or BiP [18]. We found that the transformed strains expressing either the -KDEL or the -HDEL hPDI, secreted the protein into the