The linear (1-+ 6)-p-D-glucans pustulan and luteose were effective competitive inhibitors of killer toxin action. Affinity chromatography of killer toxin on a pustulan-Sepharose column showed that toxin bound directly to a (1 6)-plinked polysaccharide. Other polysaccharides found in yeast cell walls, including (1-3)-p-D-glucan, mannan, chitin, and glycogen, were not effective as inhibitors of toxin. Fractionation of yeast cell walls was attempted to identify the toxin receptor in sensitive Saccharomyces cerevisiae. The receptor activity was retained among the insoluble glucans in alkali-washed cells; yeast mannan and alkali-soluble glucan had little receptor activity. A minor fraction of receptor activity was removed from alkali-washed cells by hot acetic acid extraction, a procedure which solubilized some (1-* 6)-3-D-glucan and glycogen. The major fraction (>70%o) of receptor activity remained with the acid-insoluble (1 6)-+and (1-3)-p-glucans. Zymolyase, an endo-(1-3)-3-D-glucanase, solubilized a substantial fraction of the receptor activity in the acid-insoluble glucans. The receptor activity in yeast cell walls was periodate and (1-* 6)-p-D-glucanase sensitive, but was resistant to (1-. 3)-p-D-glucanase and a-amylase. The acidsoluble glucan fractions of a sensitive strain and a krel-I receptor-defective toxinresistant mutant were examined. The krel-l strain had a reduced amount (ca. 50%) of (1-. 6)-3-D-glucan compared with the sensitive parent strain. A sensitive revertant of the krel-I strain regained the parental level of glucan. These results implicate (1-. 6)-3-D-glucan as a component of the yeast cell wall receptor for killer toxin.
3S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes krel or kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant krel-1. This implied that binding to krel-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 106 M-' and a receptor number of 1.1 x 107 per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 3S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from krel-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 107 per cell) wall receptor appeared necessary for toxin action, as few as 2.8 x 104 toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics of killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.
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