The pGKL1 Encoded Zymocin a Biogenesis

The K. lactis zymocin is a heterotrimeric (a, P, y) glycoprotein, the subunits of which display molecular masses of 99, 30, and 28 kDa, respectively (Stark and Boyd 1986). The smallest subunit (y) is encoded by ORF4 of pGKL1; the two larger ones originate from a single gene product (Orf2p) by posttrans-lational processing (Hishinuma et al. 1984; Stark et al. 1984, 1990; Sor and Fukuhara 1985; Stark and Boyd 1986; Tokunaga et al. 1987).

Both Orf2p and Orf4p carry typical signal peptides mediating their entry into the secretory pathway. Upon translocation to the endoplasmic reticulum (ER), signal peptides are probably cleaved off (Tokunaga et al. 1990); subsequently, in the Golgi, processing of Orf2p at positions KR29 and KR894 occurs by means of the Kex1 protease, ultimately giving rise to mature a-and P-subunits (Stark and Boyd 1986; Wesolowski-Louvel et al. 1988; Tanguy-Rougeau et al. 1988; Stark et al. 1990). Secreted holotoxin forms the a,P,y-complex in which P and y are covalently linked via a disulfide bond; the a-subunit at least contains one internal disulfide bridge (Stark and Boyd 1986; Stark et al. 1990). Protein glycosylation is restricted to the a-subunit, in which a single N-linked oligosaccharide chain was detected, whereas both P and y are non-glycosylated (Stark and Boyd 1986). Inhibition of either a-subunit glycosylation by tunicamycin or precursor processing by kex1 mutation pre vents zymocin secretion (Sugisaki et al. 1985; Wesolowski-Louvel et al. 1988; Tanguy-Rougeau et al. 1988). The y-subunit, though carrying a functional signal peptide shown to efficiently mediate secretion of heterologous proteins, fails to be efficiently secreted in the absence of the aP-precursor protein (Tokunaga et al. 1988, 1989, 1990, 1991). Thus, zymocin y secretion apparently requires cosecretion and interaction with the glycosylated a-subunit or the aP-precursor (Tokunaga et al. 1990). Interestingly, the y-subunit is efficiently secreted without the need for the a^-precursor when the heterologous pre-pro sequence of the S. cerevisiae a-mating-type factor is placed in between the y-signal peptide and the mature polypeptide (Tokunaga et al. 1989, 1990, 1992). Since the pre-pro sequence is cleaved by the KEX protease in the Golgi and basal secretion levels of the native y-subunit are not affected by a kex mutation, it has been assumed that both unprocessed pre-pro sequence and aP-precursor govern y passage from the ER to the Golgi (Tokunaga et al. 1990; Gunge and Tokunaga 2004). Hence, even though y secretion requires cosecretion of toxin subunits, the assembly of holotoxin is not essential and interaction between separately encoded subunits (i.e., y and aP) most likely occurs prior to cleavage of the aP-precursor into mature a- and P-subunits (Tokunaga et al. 1990).

The zymocin encoding killer plasmids pGKL1 and pGKL2 have been transferred to other yeast species, such as Kluyveromyces marxianus (synonyms K. fragilis and Candida pseudotropicalis) and S. cerevisiae (Gunge and Sak-aguchi 1981; Gunge et al. 1982; Sugisaki et al. 1985). Irrespective of their host they confer the ability to secrete zymocin, suggesting toxin biogenesis to be similar. Indeed, S. cerevisiae cells harboring pGKL1 and pGKL2 were shown to secrete zymocin consisting of correctly processed subunits. Moreover, mutations in the KEX2 gene, which is homologous to K. lactis KEX1, prevent secretion of both holotoxin and the aP-precursor (Stark et al. 1990; Tokunaga et al. 1990).

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