Identification of tonoplast and plasma membrane in membrane fractions from garden cress (Lepidium sativum L.) with and without filipin treatment

Membranes from roots of Lepidium sativum L. were investigated in situ and after fractionation by applying morphological and biochemical methods. After freeze-fracture combined with filipin labelling the tonoplast and the plasma membrane could be easily characterized by the frequency of intramembranous particles and the arrangement of filipin-induced lesions. On tonoplast vesicles, the filipin-induced lesions were arranged in clusters of different size whereas they were evenly distributed on plasma membrane vesicles. Enrichment of tonoplast and plasma membrane in different fractions was documented by filipin labelling, phosphotungstic acid staining and by the profiles of marker enzyme activities and ATP-dependent H⁺-transport. Additionally, the presence of rightside-out and inside-out vesicles of both tonoplast and plasma membrane could be demonstrated. It was found that filipin labelling used in combination with freeze-fracturing is suitable for quantitative determinations of the percentages of tonoplast and plasma membrane in membrane fractions, which have been found to be more than 40% for the tonoplast and about 40% for plasma membrane in the respective enriched fractions.Abbreviations EF extraplasmatic fracture face - FIL filipin induced lesion - IMP intramembranous particle - PF plasmatic fracture face - PTA phosphotungstic acid-chromic acid stain - UDPG uridine 5-diphosphate glucose A preliminary report was presented at the joint Annual Meeting of the Belgian and German Societies for Cell Biology, Bonn, March 1985Dedicated to Professor Augustin Betz on the occasion of his 66th birthday