A novel aspartyl protease allowing KEX2‐independent MFα propheromone processing in yeast

Abstract

Mutants of Saccharomyces cerevisiae which lack the KEX2-encoded endopeptidase are unable to process proteolytically the mating factor alpha (MF alpha) propheromone produced from the chromosomal MF alpha 1 and MF alpha 2 genes (Julius et al., 1983). Overproduction of pheromone precursor from multiple, plasmid-borne MF alpha genes did, however, lead to the production of active MF alpha peptides in the absence of the KEX2 gene product. S. cerevisiae therefore must possess an alternative processing enzyme. The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase. To identify the gene responsible for the alternative processing, we have isolated clones which allowed production of mature MF alpha in a kex2-disrupted strain even from the chromosomal MF alpha genes. The gene isolated in this way was shown also to be essential for the KEX2-independent processing of propheromone overproduced from plasmid-borne MF alpha 1. The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BAR1 gene products from S. cerevisiae. In contrast to the BAR1 gene product, the novel aspartyl protease (YAP3 for Yeast Aspartyl Protease 3) contains a C-terminal serine/threonine-rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product. The corresponding gene YAP3 was located to chromosome XII. The normal physiological role of the YAP3 gene product is not known. Strains disrupted in YAP3 are both viable and able to process the mating factor a precursor.

Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae

“…Escherichia coli DH5α [F − recA1 endA1 gyrA96 hsdR17 supE44 relA ∆(argF-lacZYA) U169 φ80dlacZ∆M15 λ − ] was used as a general host for the amplification and propagation of all constructed plasmids. The following yeast (Saccharomyces cerevisiae) strains were used throughout this study : MS300c [MATα leu2 ura3-52 ski2-2 (K28 killer strain)] (Schmitt & Tipper, 1990) ;192.2d (MATα ura3 leu2) (Schmitt et al, 1996) ; SEY6210 (Eisfeld et al, 2000) ; HKY20-11A 112 trp1-1 ura3-1 ∆yap3\yps1 ::LEU2) (Fuller et al, 1989) ; BFY113 (MATa ura3 ade2 trp1 leu2 his3 can1 kex2 ::TRP1) ; W303-1B (MATa ura3 ade2 trp1 leu2 his3 can1) ; and ME938 (MATα bar1 leu2-3,112 gal2 ura3 his3 kex2 ::ura3 yap3\yps1 ::LEU2) (Egel-Mitani et al, 1990). All yeast cultures were grown at 30 mC either in complex YEPD medium or in synthetic medium (YNB) supplemented with the appropriate amino acid\base requirements of each strain.…”

“…In yeast, Yps1p (formerly called Yap3p) is another membrane-anchored aspartyl protease of the yapsin family, which has been shown to be responsible for presomatostatin cleavage after LeuGlu-Arg in recombinant somatostatin expressing yeast cells (Bourbonnais et al, 1993). Furthermore, overexpression of YAP3\YPS1 in a ∆kex2 null mutant can partially suppress loss of Kex2p activity (Egel-Mitani et al, 1990), indicating some overlap in cleavage specificity between Kex2p and Yps1p. Interestingly, the predicted endopeptidase cleavage site upstream of the toxin's α N terminus (Leu-Glu-Glu-Arg%*) shows striking similarity to the sequence Leu-Glu-Arg recognized during presomatostatin processing in yeast, and the yapsin Yps1p might indeed play a role in the maturation of the α N terminus.…”

Amino Acid Sequence Requirement for Efficient Incorporation of Glycosylphosphatidylinositol-associated Proteins into the Cell Wall of Saccharomyces cerevisiae

“…A 3ϫ HA epitope sequence was inserted in-frame into the 514-position of the Yap3p amino acid sequence (30). A fragment containing the 400 base pairs of the 5Ј-noncoding region and the N-terminal 514 amino acids of Yap3p was amplified by PCR and inserted into the SphI and SalI sites of pEHA, generating pEHAnYAP3.…”

Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2′ positions

“…In S. cerevisiae t~e KEX2 gene encodes a serine protease which processes proct factor and pro-killer toxin during their transit through the secretory pathway (for reviews see [1][2][3]). A second non-essential gene encodes another potential processing enzyme, Yap3 Ca east aspartic protease 3), a 569 residue protein capable of p~ocessing pro-et-mating factor when the propheromone is o',erexpressed in KEX2 deficient S. cerevisiae [4]. Recently a ttird novel proprotein processing aspartic protease, MKC7, has been cloned from S. cerevisiae [5].…”