# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action
### Secondary Receptors for P. membranifaciens Killer Toxins  
It has been described that, for most toxins, a second step that implies toxin transfer to the plasma membrane and subsequent bind to the secondary receptor mediates killing. The number of studies conducted for the determination of the presence and description of secondary receptors are scarce. However, the evidences accumulated during years of investigation about the effects of killer toxins on sensitive yeast cells corroborated that, in addition to primary receptors, specific and essential secondary receptors should be involved in the killing mechanism. In the case of K1 toxin, the most studied killer toxin, the resistance of whole cells of _kre_ mutants to K1 and the sensitivity of the spheroplasts of _kre_ mutants and wild-type cells, indicate the existence of these secondary receptors [98; 111]. K1 toxin, after its binding to cell wall (1\(\rightarrow\)6)-\(\beta\)-D-glucans [85], interacts with Kre1p, an \(O\)-glycosylated GPI-anchored cell wall protein. Mutational analyses reveal that solely \(\alpha\) subunit interacts with Kre1p. \(\Delta\)_KRE1_ mutants show complete resistance to K1, indicating that Kre1p acts as a receptor in the cytoplasmic membrane [21; 98; 103; 112; 99]. In addition, a few receptors for other toxins have been described. The H/KDEL receptor Erd2p mediates K28 toxin binding and uptake in yeast spheroplasts, a viral A/B toxin bearing an HDEL motif at the \(\beta\)-subunit. In vivo toxicity to K28 depends on the existence of Erd2p [113]. In the case of _Kluyveromyces lactis_ zymocin, Ipt1p seems to be involved as a membrane receptor. _KT16_, which is allelic to _IPT1_ (coding for mannosyl-dinositolphospho-ceramide [M(IP)2C] synthase), join in a step that follows recognition of the primary receptor (chitin) of the toxin but precedes the role of the multi-subunit Elongator, involved in histone acetylation and transcription, the most probable toxin target [114]. Speaking specifically about _Pichia_ toxins, only the membrane receptor for the PMKT toxin has been described. Some unidentified receptors have been hypothesized for other _Pichia_ toxins, in an attempt to explain the binding capability and sensitivity of toxin-treated spheroplasts [115; 116]. PMKT resembles K1 mechanism of action, using a similar strategy to reach the cytoplasmic membrane, involving a two-step interaction with the primary receptors (1\(\rightarrow\)6)-\(\beta\)-D-glucans) and cell wall-localized secondary receptors which are also plasma membrane-associated. PMKT utilizes as the secondary membrane receptor a GPI-anchored protein of the cell wall [76]. This protein, called Cwp2p, can be purified using PMKT-affinity chromatography, indicating the existence of an in vitro interaction between both proteins. It was found, in a collection of 288 viable deletion mutants for proteins related to the cell periphery, as appeared in the CYGD (Comprehensive Yeast Genome Database), that 15 ORFs increased protoplast resistance to the toxin when deleted. This agrees with the hypothesis that Cwp2p is a secondary receptor for PMKT. Furthermore, complete yeast cells and _cwp2\(\Delta\)_ protoplasts are resistant and also unable to bind PMKT, demonstrating once again that Cwp2p is necessary for PMKT toxicity. In addition, it should be indicated that, in its definitive state, Cwp2p is bind to (1\(\rightarrow\)6)-\(\beta\)-d-glucans and this is the most probable way by which PMKT interact closely with Cwp2p, the plasma membrane receptor located in the cell wall. It has been also demonstrated, using a collection of deletion mutants related to GPI (Glycosyl Phosphatidyl Inositol) anchor biosynthesis, that Cwp2p is present in both cellular locations the plasma membrane and cell wall, as usually occur for most GPI-proteins of the yeast cell wall [76; 98].