S. Beggah et al., Intra- and intermolecular events direct the propeptide-mediated maturationof the Candida albicans secreted aspartic proteinase Sap1p, MICROBIO-UK, 146, 2000, pp. 2765-2773
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.