Orientation and Integrity of Plasma Membrane Vesicles Obtained from Carrot Protoplasts

Abstract

Two fractions enriched in plasma membrane derived from suspensioncultured 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(Cconcanavalin A to purified membrane fractions, and the competitive removal of lacetyl-"Clconcanavalin A from purified membranes derived from lacetyl-4"Cconcanavalin 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 membraneenriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.Membrane preparations of high purity and known physical orientation (sidedness) have been extremely valuable in the elucidation of membrane properties in various non-plant systems (10,14,31). Knowledge of the purity and orientation of plasma membrane vesicles is critical for proper interpretation of transport and other biochemical activities of the plasma membrane such as cell wall synthesis. Relatively pure plant plasma membrane preparations have been obtained from a number ofsources (2,12,13,21,25). In only one instance (2) was the protein composition of the plasma membrane fraction (as determined by electrophoretic analysis) clearly distinguishable from a mitochondria-enriched fraction, but even this plasma membraneenriched fraction did not appear to be significantly different from other fractions enriched in Golgi, ER, and possibly tonoplast. This was attributed to the plasma membrane being a significant contaminant in the other fractions. Although a preparation may be primarily plasma membrane as measured by PACP3 staining (29), the protein composition as determined by electrophoretic

Mg2+‐ATPase activity in wheat root plasma membrane vesicles: Time‐dependence and effect of sucrose and detergents

“…Thus, the detergent Triton X-100 is now used to determine the sidedness of plasma membrane vesicles (e.g. Randall and Ruesink 1983, Larsson et al 1984, Korner et al 1985, Berczi and M0ller 1986, Hodges and Mills 1986, Yoshida et al, 1986, The assumptions inherent in such an approach will be discussed later.…”

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers

“…Additionally, limited 'attention to markers of other intracellular organelles which may contaminate plasma membrane fractions has compromised the degree of purity of many preparations. Finally, reports referring to the orientation of plasma membrane vesicle preparations obtained by centrifugation methods only describe the presence of mixed populations of right-side-out and inside-out vesicles (Monk et al, 1989;Randall & Ruesink, 1983).…”

Two-dimensional gel electrophoresis of membrane proteins from ectomycorrhizal fungi

“…Ultrastructural studies have shown that the surface area of the plasma membrane and endoplasmic reticulum increases extensively in the ectomycorrhizal symbionts (Massicotte et al, 1987;Kottke and Oberwinkler, 1989). This increase is presumably associated with recognition, nutrient trafficking, and secreted protein biosynthesis (Smith and Smith, 1990;Martin and Hilbert, 1991 (Dupont and Leonard, 1980;Randall and Ruesink, 1983 (1983). P tinctorius was grown on Pachlewski's medium in 2% agar (Martin et al, 1990) and C geophilum was grown in liguid culture in Pachlewski's medium (Martin et al, 1983 (1990) and Martin et al (1991).…”