Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Abstract

b Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of ␤-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-␤-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the ␤-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ␤-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.T he production of antimycotic killer toxins has been observed in several yeast genera and proved to be a widespread phenomenon (1, 2). Killer strains of Saccharomyces cerevisiae secrete protein toxins derived from a family of double-stranded RNAs (dsRNAs). The toxins have been grouped into four types (K1, K2, K28, and Klus) based on their killing profiles and lack of crossimmunity (3, 4). Such proteins are able to kill the nonkiller yeast, as well as yeast of other killer types, while the toxin-producing cells remain immune to their own or to the same type of killers (4, 5). K1 toxin disrupts the regulated ion flux across the plasma membrane, leading to the death of sensitive yeast strains (6, 7). The killing action of K1 toxin involves at least two steps. During the first step, the toxin binds to the cell wall, whereas the second step leads to the translocation and insertion of the toxin into the plasma membrane (6). Beta-1,6-glucan was originally proposed to be a cell wall receptor for K1 (8). Analysis of several kre mutants demonstrated that decrease of the cell wall ␤-1,6-glucan level leads to K1 resistance, thus confirming the involvement of this type of glucan in toxin binding (9). During the second step, K1 toxin interacts with plasma membrane receptors and disrupts the functional integrity of the plasma membrane either by inducing the formation of new ion channels (7) or through the activation of existing potassium channels (10). Products of TOK1 (protein, forming the potassium ion channel) and KRE1 (glycoprotein, involved in ␤-glucan assembly) have be...

Understanding the killing mechanism of action by virus-infected yeasts

“…The analysis showed that as long as the saturation of the receptors on the cell wall was avoided, the binding kinetics remained constant (Fig, 1b). Similar binding rates were reported in experiments where the initial unbound toxin concentration was varied by more than 50-fold, providing further support for these findings [25,27].…”

“…K1 molecules/cell was proposed[27]. The number of β -1,6-D-glucans on a haploid parent yeast cell wall was estimated to vary in the range of 6,600,000 to 11,000,000[26,53], and would be even higher for diploid cells than these reported here.…”

“…Cells that are susceptible to these toxins were identified to possess two different types of sites or receptors that bind the killer toxin with different affinities [25]. The first step of K1/K2 binding was reported as the low affinity, high velocity, energy independent adsorption of the killer toxin on β -1,6-D-glucans embedded in the cell wall [26,27]. Once bound, the toxin had a high affinity, low velocity, energy-dependent interaction with Kre1p receptors; the glycosyl-phosphatidylinositol-linked glycoproteins located on the cell membrane [28,29].…”

Evidence for Proinflammatory β-1,6 Glucans in the Pneumocystis carinii Cell Wall

“…Hydrolysis and fractionation techniques used routinely to determine the polysaccharide content of the sugar of interest have demonstrated that not even trace amounts of di-, tri-, or tetrasaccharides are observed in pustulan, indicating that the molecule is linear in nature and contains exclusive ␤-1,6 glucan linkages (21). Other researchers have routinely used pustulan to study ␤-1,6 glucan-C-type lectin receptor (CLR) interactions and yeast ␤-1,6 glucan cell wall biology (22)(23)(24)(25)(26).…”

Dynamic modelling of the killing mechanism of action by virus-infected yeasts

“…Cells that are susceptible to these toxins were identified to possess two different types of sites or receptors that bind the killer toxin with different affinities [26]. The first step of K1/K2 binding was reported as the low affinity, high velocity, energy independent adsorption of the killer toxin on b-1,6-D-glucans embedded in the cell wall [27,28]. Once bound, the toxin had a high affinity, low velocity, energydependent interaction with Kre1p receptors, which are glycosyl-phosphatidylinositol-linked glycoproteins located on the cell membrane [29,30].…”