# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action  
### Primary Receptors in Sensitive Yeast Cells  
It is widely accepted that cell surface polysaccharides act as receptors for bacteria, viruses, and toxins [52; 94]. Yeast killer toxins have different mechanisms of action but most toxins are able to kill sensitive yeasts in a two-step process that is receptor-mediated. There are two kinds of killer toxin receptors: the primary, usually located on the cell wall, and the secondary receptor, that exist on the plasma membrane of the sensitive cells. The interaction with the first receptor within the cell wall of a sensitive target cell involves a fast and energy-independent binding [20; 49; 75; 76; 95; 96; 97; 98; 99]. Cell walls are mainly composed by carbohydrates, some of them free and some linked to proteins. Overall, the main components of the yeast cell wall are (1\(\rightarrow\)3)-\(\beta\)-D-glucans, (1\(\rightarrow\)6)-\(\beta\)-D-glucans, mannoproteins and chitin, which is concentrated in the bud-scar region [100; 101].  
A diversity of cell wall receptors for killer toxins exist, evidencing that all the main cell wall components of yeasts could function as the primary receptor for a killer toxin. For example, (1\(\rightarrow\)6)-\(\beta\)-D-glucans act as cell wall receptors for K1 and K2 killer toxins of _S. cerevisiae_[44; 52], for _Hansenispora uvarum_ killer toxin [102] and for _Pichia anomala_ DBVPG3003. (1\(\rightarrow\)3)-\(\beta\)-D-glucans are primary receptors for _Pichia anomala_ NCYC434 [65], mannoproteins are receptors for KT28 of _S. cerevisiae_ and also _Zygosaccharomyces bailii_ killer toxin [63; 96; 103; 104], and, finally, chitin is the cell receptor for _Kluyveromyces lactis_ killer toxin [97]. In agreement with that, reported mutations in a gene/genes involved in the synthesis or architecture of the cell wall are observed to be resistant to killer toxins. _Kre_-Killer Resistant mutants have been obtained for K1 [105] and, in the same way, _mnn_-Mannoprotein mutants for K28 [106]. Mutational analyses revealed that both subunits of K1, \(\alpha\) and \(\beta\), are involved in glucan binding [107; 108]. It has been indicated that the primary receptors in the cell wall facilitate the primary contact of the killer toxins with the sensitive yeasts or even mediates close contacts between the toxin and secondary receptors on the plasma membrane [96].  
Using both in vivo and in vitro approaches, in previous works [75], it was confirmed the role of (1\(\rightarrow\)6)-\(\beta\)-D-glucans as primary targets for PMKT and the utility of such interaction as an effective means for PMKT purification [19]. Finally, it was shown that the adsorption of PMKT to purified (1\(\rightarrow\)6)-\(\beta\)-D-glucans occur within the first 2 min of contact, indicating, as previously hypothesized, that this process is an energy-independent binding and toxin-specific, since polysaccharides with the following linkages: (1\(\rightarrow\)3)-\(\beta\)-, (1\(\rightarrow\)4)-\(\beta\)-, (1\(\rightarrow\)4)-\(\alpha\)-, or (1\(\rightarrow\)6)-\(\alpha\)- were unable to bind PMKT.  
Yeast mannans may be covalently linked to cell wall proteins, so that the term \"mannoproteins\" adequately reflects their status in the yeast cell wall. Mannoproteins have been described as antigenic determinants, also functioning as receptors or even as support for other receptors of different nature [109; 110]. Conversely to what was found for PMKT, in a complementary study for the determination of the binding capacity of different yeast cell-wall fractions, cell wall glucan fractions do not bind PMKT2, whereas mannoproteins and mannan purified from sensitive yeasts were able to, showing the existence of a cell wall receptor of different nature for PMKT2 [20].