Membrane orientation of droplets prepared from Chara corallina internodal cells

It is generally accepted that the membrane surrounding droplets from charac ean cells originates from the tonoplast, but there is some uncertainty rega rding droplet membrane sidedness. This issue was addressed directly by comb ining two different droplet isolation methods and the patch clamp technique . Neutral red accumulation was used to demonstrate the presence of H+-trans port over the membrane and to predict membrane orientation. Two types of dr oplet populations with differently oriented membranes could be formed in an iso-osmotic bath solution. Cytoplasmic droplets (cytosolic side of the ton oplast inside) contained cytoplasm, while the second type of droplet popula tion contained vacuolar sap (vacuolar droplets, vacuolar side of the tonopl ast inside). Smaller vesicles also appeared inside the droplets, with an ap parently inversely oriented membrane. Confocal laser scanning microscopy in directly demonstrated that, at least with one of the droplet isolation meth ods, the plasma membrane entirely remains in the internodal cell after intr acellular perfusion. Both types of droplet populations allowed the formatio n of excised patches and single-channel measurements by the patch clamp tec hnique. Properties of anion channels in the tonoplast could be used to prov e the predicted membrane orientation, knowing that Ca2+ can only activate t hese channels from the cytosolic side. These results provide useful data fo r studies addressing ligand-binding, block and modulation, organization and interaction of proteins within the membrane or with other regulatory facto rs, where it is important to control membrane orientation.