Restriction of glycolipids to the outer half of a plasma membrane: Concanavalin A labeling of membrane halves in acanthamoeba castellanii

Fracture-label, a method that permits the cytochemical characterization of faces produced by freezefracture, was used to determine the partition and distribution of a glycolipid on membrane fracture faces of Acanthamoeba castellanii cells. After treatment with concanavalin A (Con A), the glycolipid (a lipophosphonoglycan, LPG) was labeled with colloidal gold coated with horseradish peroxidase. The label was abundant over exoplasmic fracture faces (face E) of plasma membranes, but absent from protoplasmic fracture faces (face P). We conclude that, in A. castellanii, glycolipid molecules are restricted to the outer half of the plasma membrane. This conclusion is confirmed by experiments with cells disrupted by freezing and thawing, where access of label to the cell interior did not result in labeling of the inner surface. Our results establish the exclusive localization of a glycolipid to the outer half of a plasma membrane. Fracture-label is proposed as a new technique to investigate the distribution and partition of glycolipids in plasma and intracellular membrane halves.