Intra- and intermolecular events direct the propeptide-mediated maturationof the Candida albicans secreted aspartic proteinase Sap1p

Pathogenic yeasts of the genus Candida secrete aspartic proteinases (Sap) w hich are synthesized as preproenzymes. Expression of the C. albicans SAP1 g ene lacking the propeptide-coding region in the methylotrophic yeast Pichia pastoris does not lead to the secretion of the enzyme into the culture sup ernatant, but results in an accumulation of recombinant protein in the cell . Go-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion. A deletion analysis reve aled that only a 12 aa sequence in the propeptide, corresponding to a highl y conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant. No Sap1p was secret ed when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence. however, the simultaneous production of equ ivalent amounts of Sap1p and his-tagged Sap1p (H-6-Sap1p) with a mutated an d a non-mutated propeptide, respectively, led to the secretion of both prot eins in a ratio of approximately 1:2. The restoration of Sap1p secretion oc curred at the expense of secretion of H-6-Sap1p since the total activity wa s comparable to that of strains producing only H-6-Sap1p with a non-mutated propeptide. In contrast, the proteolytic activity of strains secreting Sap 1p and H-6-Sap1p both with a functional propeptide was twice that of strain s producing either Sap1p or H-6-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant Altogether, these resul ts show that the propeptide can only function once and that the maturation of recombinant C. albicans secreted aspartic proteinase Sap1p is directed t hrough a combination of intra- and inter-molecular pathways.