CLONING, NUCLEOTIDE-SEQUENCE AND FUNCTIONS OF XPR6, WHICH CODES FOR ADIBASIC PROCESSING ENDOPROTEASE FROM THE YEAST YARROWIA-LIPOLYTICA

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not bee n cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced abili ty to cleave after Lys-Arg of an artificial substrate. The XPR6 gene w as cloned by complementation by screening for restoration of productio n of alkaline protease activity. Sequencing of a 3735 base pair SalI-S phI XPR6 fragment revealed a large open reading frame with a coding ca pacity of 976 amino acids (molecular weight, 110 016). The deduced ami no acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic ami no acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline e xtracellular protease was produced indicating that the low levels prod uced 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. Cel ls remained physically attached after budding and continued to bud for ming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aber rations were largely eliminated by growing cells in medium at pH 4. Fo urth, no mycelial forms were observed regardless of the pH.