Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

Abstract

PMKT is a channel-forming killer toxin secreted byPichia membranifaciens. To identify novel genes thatmediate cellular resistance to PMKT we screened acollection of 288 deletion mutants. We found 29 openreading frames (ORFs) that, when deleted, conferresistance to PMKT. In addition, the deletion of 15ORFs was observed to increase protoplast resis-tance, in agreement with the initial assumption that aplasma membrane receptor for PMKT exists. Wholecells and protoplasts of a cwp2D mutant were foundto be completely resistant to PMKT and were unableto bind PMKT, indicating that Cwp2p interacts with it.A protein with a molecular mass of 11.7 kDa was puri-fied from PMKT-affinity columns. This protein wassequenced and identified as Cwp2p. Glycosylphos-phatidylinositol (GPI) anchoring-defective mutantswere much less sensitive to PMKT, as were wild-typeprotoplasts pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchoredproteins, indicating that the GPI-anchored precursorof Cwp2p is also necessary for PMKT activity.Carboxyfluorescein-entrapped liposomes containinga purified GFP–Cwp2p fusion protein in their mem-branes were much more sensitive to PMKT thanprotein-free liposomes. Cwp2p and its GPI-anchoredprecursor are proposed for the first time to beinvolved as PMKT secondary receptors.