The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells

Examination of organelle- and membrane-specific processes such as signal transduction necessitates the use of plasma membrane vesicles with cytoplasmic side-in orientation. we are interested in the structural identity and subcellular localization of in vivo [³²P]phosphoric acid ([³²Pi])-labeled phosphoinositides, including the recently discovered phosphatidyl-scyllo-inositol, for signal transduction studies. In the first part of this investigation, plasma membrane vesicles from barley aleurone cells were isolated employing the aqueous polymer (Dextran and polyethylene glycol) two-phase partition method. The membrane vesicles that partitioned into the upper and lower phases of the aqueous polymer two-phase system were characterized and the purity of the vesicles ascertained by assaying for two marker enzymes, K⁺-stimulated, Mg²⁺-dependent adenosine triphosphatase (EC 3.6.1.3, ATPase), localized in the plasma membranes, and cytochrome c oxidase, localized in the mitochondria. Inhibitors for ATPases such as azide, molybdate, and vanadate were used to distinguish between plasma membrane-associated and intracellular membrane-as-sociated ATPases. These inhibitor studies suggest that the plasma membrane preparation contained about 7% of intracellular membrane vesicles and the intracellular membrane fraction contained about 6% of plasma membrane vesicles. Orientation of the plasma membrane vesicles was ascertained by measuring the latent ATPase activity. These latency studies suggest that about 95% of the plasma membrane vesicles were of cytoplasmic side-in orientation. In the second part of this investigation, intracellular distribution and in vivo [³²Pi] labeling of phosphoinositides in the plasma membranes and intracellular membranes were investigated. Preferential accumulation of [³²Pi]-labeled phosphatidyl-myo-inositol monophosphate (myo-PIP) and phosphatidyl-myo-inositol bisphosphate (myo-PIP₂) was observed in the plasma membrane. However, scyllo-phosphatidylinositol (scyllo-PI) was detected in both the plasma membrane and the intracellular membranes. The cellular concentration of myo-phosphoinositides was determined, and, after 24 h of labeling with [³²Pi], the ratio of radiolabel in myo-PI, PIP, and PIP₂ paralleled the relative concentrations in aleurone cells.