Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor

Abstract

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.

Mannosyl-Diinositolphospho-Ceramide, the Major Yeast Plasma Membrane Sphingolipid, Governs Toxicity of Kluyveromyces lactis Zymocin

“…Consistent with an impact on early events, class I mutations in KTI2/CHS3 and KTI10/ PMA1 protect solely against exozymocin and affect chitin synthesis and plasma membrane H ϩ -ATPase function, respectively (11,33,43). In line with this, holozymocin binds chitin in vitro and has exochitinase activity (10,33).…”

“…Consistent with an impact on early events, class I mutations in KTI2/CHS3 and KTI10/ PMA1 protect solely against exozymocin and affect chitin synthesis and plasma membrane H ϩ -ATPase function, respectively (11,33,43). In line with this, holozymocin binds chitin in vitro and has exochitinase activity (10,33). As judged from the finding that hygromycin B, whose antibiotic action requires an intact membrane potential established by H ϩ -ATPase Pma1/ Kti10, is inert against kti10 cells, plasma membrane energization is likely to be required for zymocin action (43,50).…”

“…Combining kti10 and kti6 mutations involved yeast matings between ARB68 (kti10) and ARBK10 (kti6-3) ( Table 1) or PCR disruption of KTI6 by the K. lactis LEU2 gene (see below) using previously described protocols (33). Sphingolipid analysis essentially followed the protocol for yeast radiolabeling with [ 3 H]myoinositol followed by 5% (vol/vol) trichloroacetic acid extraction (14).…”

“…As judged from the finding that hygromycin B, whose antibiotic action requires an intact membrane potential established by H ϩ -ATPase Pma1/ Kti10, is inert against kti10 cells, plasma membrane energization is likely to be required for zymocin action (43,50). Given the strongly hydrophobic nature of the ␤-subunit, to which the active ␥-subunit is linked via a disulfide bond, a tentative picture that emerges for early zymocin-induced events includes chitin docking followed by delivery of the ␥-toxin subunit to the membrane and subsequent subcellular import (33,43,56).…”

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

“…In the search for novel and more selective antifungal agents, fungal cell wall components represent primary targets, as these structures are restricted to yeasts and higher fungi and do not occur in human cells [44,45]. Each of the mentioned cell wall components has been shown to act as the primary binding site for different yeast killer toxins [46][47][48][49]. In addition, for killer toxins secreted by various strains of the yeast Hansenula it was demonstrated that these toxins not only bind to yeast cell wall components, but that they also strongly inhibit de novo L-1,3-D-glucan biosynthesis [50,51].…”