Orientation and integrity of plasma membrane vesicles obtained from carrot protoplasts

Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-(14)C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-(14)C]concanavalin A from purified membranes derived from [acetyl-(14)C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of ;tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly ;leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be ;leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.