Activation and Processing of Non-anchored Yapsin 1 (Yap3p)

Abstract

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel subclass of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding ϳ1 g of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala 68 . The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated ␣ and ␤, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at ϳ4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

“…We expressed an N-terminal HA-tagged version of Yps1 ( HA Yps1) in WT and pmt2 mutant strains and analyzed its glycosylation by Western blot before and after deglycosylation using Endo H. In WT, HA Yps1 showed an apparent molecular mass of ϳ90 kDa that shifted to ϳ75 kDa upon Endo H treatment (Fig. 7, lanes 1 and 3), indicating moderate N-glycosylation consistent with previous data (39). In the pmt2 mutant, molecular mass and abundance of the ϳ90 kDa form of HA Yps1 slightly decreased, and in turn a prominent high molecular mass form (Ͼ270 kDa) appeared (Fig.…”

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

“…In addition to a Kex2 homolog, the aspartic yapsin proteases (18,30,36) could also be involved, since their substrate specificities overlap those of Kex2 in S. cerevisiae (10). Yps1 (yapsin 1, previously termed Yap3; EC 3.4.23.41) is the best-characterized yapsin and is primarily active at the plasma membrane but is also found in the extracellular medium and transiently active in the late secretory pathway (2,14,28). It has frequently been implicated in the degradation of secreted heterologous proteins at basic amino acids in S. cerevisiae (see, for example, reference 28).…”

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin

“…Gene overexpression in the native organism was also revealed to be effective for the production of various secreted proteases such as A. niger prolyl endopeptidase AN-PEP, S. cerevisiae barrier pepsin (Bar1), Saccharomyces cerevisiae yapsin 1 (Yps1) and eight of the Candida albicans secreted aspartic proteases for further purification and biochemical characterization (Cawley et al, 1998;Edens et al, 2005;MacKay et al, 1988;Staib et al, 2008). An advantage of gene overexpression in the native organism is that the production of recombinant protein can be obtained from genomic DNA and is not obligatory from cDNA as the introns are naturally spliced from transcribed RNA.…”