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Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

Masami Hayashi1, Yukiko Shimada, Mitsushi Inomata

  • 1Cellular Signaling Group, Research Team for Functional Genomics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.

Biochemical and Biophysical Research Communications
|November 7, 2006
PubMed

Insights

Cholesterol-rich microdomains exist in the inner plasma membrane. Perfringolysin O

Area of Science:

  • Cell Biology
  • Biochemistry
  • Membrane Biology

Background:

  • Cholesterol is crucial for cell membrane function.
  • Cholesterol-rich microdomains, or lipid rafts, are important cellular structures.
  • The location of these microdomains in the inner plasma membrane leaflet remains debated.

Purpose of the Study:

  • To investigate the presence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.
  • To determine if perfringolysin O's C-terminal domain (D4) can serve as a probe for these microdomains.

Main Methods:

  • Expressed EGFP-D4 fusion protein in mouse embryonic fibroblast (MEF) cells.
  • Utilized cell fractionation and ultracentrifugation to isolate membrane raft fractions.
  • Depleted membrane cholesterol using beta-cyclodextrin to assess D4 binding specificity.
  • Subfractionated raft components to distinguish between plasma and intracellular membranes.

Main Results:

  • Over half of the expressed EGFP-D4 localized to membrane raft fractions.
  • Cholesterol depletion significantly reduced EGFP-D4 binding to raft fractions.
  • Further subfractionation indicated EGFP-D4 predominantly associates with the plasma membrane.

Conclusions:

  • The results provide strong evidence for the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.
  • Perfringolysin O's D4 domain is a reliable marker for detecting these specific membrane domains.

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