Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

Abstract

SummaryPMKT is a channel-forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2D mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT-affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring-defective mutants were much less sensitive to PMKT, as were wild-type protoplasts pretreated with phosphatidylinositolspecific phospholipase C to remove GPI-anchored proteins, indicating that the GPI-anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein-entrapped liposomes containing a purified GFP-Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein-free liposomes. Cwp2p and its GPI-anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis

“…Pichia membranifaciens CYC 1086 was the killer toxin producer used in this study (Complutense Yeast Collection, Complutense University of Madrid, Spain), originally isolated from olive brines (Marquina et al, 1992), identified according to conventional methods used in yeast taxonomy and deposited at the Portuguese Yeast Culture Collection (PYCC, Caparica, Portugal). The killer toxin from P. membranifaciens CYC 1086 (named PMKT2) was compared in the present study with PMKT obtained from P. membranifaciens CYC 1106 (Santos & Marquina, 2004a;Santos et al, 2005Santos et al, , 2007. The sensitive strain used for routine killer assays was Candida boidinii IGC 3430 (PYCC), originally isolated from olive brines.…”

“…PaT, the killer toxin produced by P. acaciae, has a tRNase activity ( Recently, it has been shown that P. membranifaciens produces a killer toxin (PMKT) that is active on spoilage yeasts and fungi (Santos et al, 2000(Santos et al, , 2004. PMKT has extensive activity against different micro-organisms but only under restricted conditions, and therefore is of interest for its potential application as an antimicrobial agent, but only in permissive environments (Llorente et al, 1997;Santos & Marquina, 2004a).Previous biochemical studies indicated that PMKT is an 18 kDa protein that interacts with the cell wall, first with the (1A6)-b-D-glucans (Santos et al, 2000) and then with CWP2p, a GPI-anchored protein (Santos et al, 2007). Regardless of certain possible additional effects, PMKT acts by disrupting plasma membrane electrochemical gradients, leading to the death of sensitive cells (Santos & Marquina, 2004b).…”

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

“…The fps1 cells do not show phenotypes with very high resistance to S. cerevisiae K1 killer toxin and P. membranifaciens killer toxin (Page et al, 2003;Santos et al, 2007), unlike resistance to HM-1. Thus, Fps1p seems to participate in the yeastkilling action of HM-1.…”

“…The glycophosphatidylinositol (GPI)-anchored proteins Kre1p and Cwp2p were believed to be the plasma membrane receptor of S. cerevisiae K1 killer toxin and PMKT respectively (Breinig et al, 2002;Santos et al, 2007). It was also reported that deletion mutants of KRE1 and CWP2 genes showed strong resistance against K1 killer toxin and PMKT respectively (Breinig et al, 2002;Page et al, 2003;Santos et al, 2007). Thus to identify the plasma membrane receptor of HM-1, HmBP, we checked the HM-1-resistance of deletion mutants of GPIanchored proteins and yeast plasma membrane proteins (these 366 genes are listed in Table S in Figure 5.…”

“…They concluded that these genes having specific cellular functions are important for the yeast-killing action of K1 toxin. Santos et al found the plasma membrane receptor of Pichia membranifaciens killer toxin (PMKT) by screening deletion mutants for resistance to the toxin (Santos et al, 2007).…”