D Toxin Immunity

Essentially, zymocin producing cells are resistant toward the toxin; such self-immunity strictly relies on the availability of the killer plasmid pair pGKL1 and pGKL2; however, partially cured strains carrying exclusively pGKL2 are susceptible to toxin (Gunge et al. 1981; Niwa et al. 1981). Accordingly, immunity is accomplished by pGKL1. As already outlined, three of the four pGKL1 genes are either involved in replication (ORF1) or encode toxin sub-units (ORF2 and ORF4). Since deletion of a pGKL1 region spanning ORF2, ORF3, and ORF4 gave rise to nonimmune nonkillers, whereas deletion solely of ORF2 produced immune nonkillers, pGKL1 ORF3 was held responsible for zymocin immunity (Hishinuma et al. 1984; Sor and Fukuhara 1985; Stark and Boyd 1986; Stark et al. 1990). In fact, expression of ORF3 from an ARS vector in a strain devoid of pGKL1 conferred toxin resistance (Tokunaga et al. 1987). However, the acquired immunity was only partial; high toxin doses (400 ng ml-1) still caused noticeable growth inhibition, and were indeed tolerated by cells carrying pGKL1. Moreover, expression of ORF3 from an ARS vector conferred immunity exclusively in the presence of pGKL2 (Tokunaga et al. 1987). Thus, it was suggested that pGKL2 mediates expression of ORF3 from ARS vectors; however, this lags behind pGKL1 based expression (Tokunaga et al. 1987; Stark et al. 1990). Alternatively, pGKL2 might encode an additional factor required for full zymocin immunity. There is an additional nonessential ORF on pGKL2 that does not exist in other yeast autonomous elements (ORF1; see above). Though the predicted polypeptide displays significant similarities to pGKL1 Orf3p this ORF has evidently no function in zymocin immunity, as deletion mutants remained immune (Schaffrath et al. 1992).

The biochemical basis of immunity so far remains unknown; nevertheless, pGKL1 ORF3 not only protect cells from exogenously applied zymocin but also antagonizes intracellularly expressed y (Tokunaga et al. 1989), indicating that zymocin protection is not realized by exclusion of y but rather involves intracellular interference with y function.

For colicin D, which exerts a zymocin-like toxic principle (see above), it was proven that the corresponding immunity protein (ImmD) structurally mimics the respective tRNA substrate and, forming a tight complex with the toxic domain, blocks the tRNase active site (Graille et al. 2004). Whether similarities between zymocin and colicins are restricted to (presumably con-vergently evolved) toxic principles or extend to like immunity mechanisms needs to be discovered.

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