The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G 1 phase of the cell cycle. Despite its heterotrimeric (abc) structure, intracellular expression of its smallest subunit, the c-toxin, is alone responsible for the G 1 arrest. The a subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3D), blocked export (chs7D) or reduced activation (chs4D), combined with mislocalization (chs4Dchs5D; chs4Dchs6D; chs4Dchs5Dchs6D) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

The toxin produced by Pichia acaciae was purified and its properties compared to those of the toxin from Kluyveromyces lactis. Like this toxin, the P. acaciae toxin is a protein comprised of three subunits (molecular masses 110,39 and 38 kDa) with an associated chitinase activity and a pH optimum between 7.0 and 7-5. P. acaciae toxin also caused G I cell cycle arrest. Of the thirteen recessive alleles that provided resistance in Saccharomyces cerevisiae to K. lactis toxin, only three also conferred resistance to P. acaciae toxin. Similarities and differences in the interactions of the two toxins with yeast cells are discussed.