Proteins are known to form functional clusters in plasma membranes. In orde
r to identify individual proteins within clusters we developed a method to
visualize by atomic force microscopy (AFM) the cytoplasmic surface of nativ
e plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-
lysine coated glass. After removal of the vitelline membrane intact oocytes
were brought in contact with coated glass and then rolled off. Inside-out
oriented plasma membrane patches left at the glass surface were first ident
ified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Mem
brane patches exhibiting the typical phospholipid bilayer height of 5 nm sh
owed multiple proteins, protruding from the inner surface of the membrane,
with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical
structures embedded in the lipid bilayer and protruding into the cytoplasm
allowed an estimation of the respective molecular masses. Proteins ranged
from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently foun
d membrane protein structure (40/ mu m(2)) had a total height of 10 nm and
an estimated molecular mass of 280 kDa.
Membrane proteins were found firmly attached to the poly-L-lysine coated gl
ass surface while the lipid bilayer was found highly mobile. We detected pr
otein structures with distinguishable subunits of still unknown identity. S
ince X. laevis oocyte is a generally accepted expression system for foreign
proteins, this method could turn out to be useful to structurally identify
specific proteins in their native environment at the molecular level. Copy
right (C) 2000 S. Karger AG, Basel.