Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells

Formation of intracellular vesicles is initiated by membrane budding. Here we test the hypothesis that the plasma membrane surface area asymmetry coul d be a driving force for vesicle formation during endocytosis. The inner la yer phospholipid number was therefore increased by adding exogenous aminoph ospholipids to living cells, which were then translocated from the outer to the inner layer of the membrane by the ubiquitous flippase. Addition of ei ther phosphatidylserine or phosphatidylethanolamine led to an enhancement o f endocytosis, showing that the observed acceleration does not depend on th e lipid polar head group. Conversely, a closely related aminophospholipid t hat is not re cognized by the flippase, lyso-alpha-phosphatidylserine, inhi bited endocytosis, and similar results were obtained with a cholesterol der ivative that also remains in the plasma membrane outer layer. Thus an incre ase of lipid concentration in the inner layer enhanced internalization, whe reas an increase of the lipid concentration in the outer layer inhibited in ternalization. These experiments suggest that transient asymmetries in lipi d concentration might contribute to the formation of endocytic vesicles.