Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Abstract

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110,016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5.5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.

Homology modelling of the catalytic domain of human furin

“…Asp46Ala, His87Ala, Asnl88Ala, Ser261Ala or Asp46Asn (Leduc et al, 1992). (Korner et al, 1991); Dm FURlDm FUR2, Drosophila melanogaster furin 1 (Roebroek et al, 1991 and furin 2 ; Ce BLIS, Caenorhabditis elegans blisterin (Peters, 1992); Ls PC2, Lymnea stagnalis PC2 (Smit et al, 1992); Hs PC2, human PC2 ; Hs PC13, human PC1/3 (Smeekens and Steiner, 1990); Mm PC4, mouse PC4 (Nakayama et el., 1992;Seidah et al, 1992); Hs PAC4, human PACE4 (Kiefer et al, 1991); Mm PC56, mouse PC5 (Lusson et al, 1993) and PC6 (Nakagawa et al, 1993); Hv PC3, Hydra vulgaris PC3-like protease (Chan et al, 1992); Y1 XPR6, Yarrowia Zipolytica XPR6 protease (Enderlin and Ogrydziak, 1994); K1 KEXl, Kluyveromyces lactis KEXl protease (Tanguy-Rougeau et al, 1988); Sc KEX2, Saccharomyces cerevisiae KEX2 protease (Mizuno et al, 1988). Residue numbering at the top corresponds to that of mature human furin (Hs FUR).…”

“…These mammalian proprotein convertases are multi-domain proteins with the serine protease (or catalytic) domain at the N-terminus (Hutton, 1990; ; they all have a specificity for cleavage after di-basic or multi-basic residues and they constitute a subfamily of the subtilisin-like serine proteinases, also abbreviated as subtilases (Siezen et al 1991). Corresponding proteases of this subfamily have now also been discovered in various eukaryotes Chan et al, 1992;Smit et al, 1992;Peters, 1992;Korner et al, 1992) including yeasts (Tanguy-Rougeau et al, 1988;Mizuno et al, 1988;Enderlin and Ogrydziak, 1994).…”

Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

“…Comparison of the signals to those of known amounts of scFv produced in E. coli as inclusion bodies (not shown) allowed us to estimate that Y28 concentration was 20 mg l −" for Y. lipolytica and 10 mg l −" for K. lactis, in shake flask culture, corresponding to 2 and 1 % of total cell proteins, respectively. (Enderlin & Ogrydziak, 1994).…”

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“…Southern blotting with a radioactive labeled probe was done as described previously (Enderlin and Ogrydziak, 1993). For low-stringency Southern blotting, washing was done at 45 C instead of at 65 C. For non-radioactive blots, DNA was blotted to a Hybond -N + membrane (Amersham) as was done for radioactive Southern blotting except that 10X SSC instead of 0·4 -NaOH was used as transfer buffer.…”