Plasma membrane biogenesis in eukaryotic cells: translocation of newly synthesized lipid

Abstract

We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag, in a linear manner up to 1 hr. Labeled sterol and phospholipids appeared in the plasma membrane, after a 30-min lag, at approximately the same rate. However, the ratio of newly synthesized sterol to phospholipid was significantly enriched in the plasma membrane relative to the whole cell, even at the earlier time points. Pulse-chase experiments indicated that sterols and phospholipids are turned over in the plasma membrane with similar, rather short half-lives. The results of these studies suggest that, although sterols and phospholipids are transported to the cell surface with similar kinetics, some sorting of the lipids must occur at an early stage in membrane biogenesis. The data are consistent with a model of lipid translocation by vesicular transport.