Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin.
PMKT 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 cwp2Delta 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 phosphatidylinositol-specific 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.
Santos
Antonio
A
Department of Microbiology, Biology Faculty, Complutense University of Madrid, 28040 Madrid, Spain.
San Mauro
Manuel
M
Abrusci
Concepción
C
Marquina
Domingo
D
D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002846:Chromatography, Affinity; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055646:Killer Factors, Yeast; D008081:Liposomes / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008970:Molecular Weight; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D010843:Pichia / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C093811:CWP2 protein, S cerevisiae; D017261:Glycosylphosphatidylinositols; D055646:Killer Factors, Yeast; D008081:Liposomes; D008562:Membrane Glycoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins