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Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

Antonio Santos1, Manuel San Mauro, Concepción Abrusci

  • 1Department of Microbiology, Biology Faculty, Complutense University of Madrid, 28040 Madrid, Spain.

Molecular Microbiology
|April 28, 2007
PubMed

Insights

Researchers identified Cwp2p as a key factor in Pichia membranifaciens killer toxin (PMKT) sensitivity. Deleting the CWP2 gene or removing GPI-anchored proteins confers resistance, suggesting Cwp2p acts as a PMKT secondary receptor.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Pichia membranifaciens secretes the channel-forming killer toxin PMKT.
  • Understanding cellular resistance mechanisms to toxins is crucial for biological and biotechnological applications.
  • Previous research suggested a plasma membrane receptor for PMKT, but its identity remained unknown.

Purpose of the Study:

  • To identify novel genes involved in cellular resistance to PMKT.
  • To elucidate the molecular mechanism of PMKT-mediated cell death.
  • To characterize the role of Cwp2p in PMKT interaction.

Main Methods:

  • Screening of a 288-deletion mutant library for PMKT resistance.
  • Characterization of PMKT binding to whole cells and protoplasts.
  • Protein purification using PMKT-affinity chromatography.
  • Mass spectrometry for protein identification.
  • Analysis of glycosylphosphatidylinositol (GPI)-anchored proteins and their role in PMKT sensitivity.
  • Liposome-based assays to assess Cwp2p function.

Main Results:

  • 29 open reading frames (ORFs) deletion mutants conferred PMKT resistance.
  • Deletion of CWP2 gene resulted in complete PMKT resistance and abolished PMKT binding.
  • Cwp2p was identified as a 11.7 kDa protein interacting with PMKT.
  • Mutants defective in GPI anchoring and wild-type protoplasts treated to remove GPI-anchored proteins showed reduced PMKT sensitivity.
  • Liposomes incorporating GFP-Cwp2p fusion protein were more sensitive to PMKT.

Conclusions:

  • Cwp2p is a novel PMKT secondary receptor.
  • The GPI-anchored precursor of Cwp2p is essential for PMKT activity.
  • Cwp2p mediates PMKT binding and subsequent cellular effects, offering new insights into toxin-host interactions.

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