Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii

Two different approaches to prepare and characterise vacuoles from the filamentous fungus Ashbya gossypii are described, i.e. the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane. By mechanical lysis of protoplasts and separation of the organelles on a stepped density gradient, we obtained a vacuolar fraction virtually free of contamination by other organelles, unlysed protoplasts and cell debris. The integrity of the isolated organelles was characterised by vital‐staining, the presence of α‐mannosidase, and retained accumulation of basic amino acids. In a second approach, the cell membrane of the fungus was selectively permeabilised by use of the saponin digitonin leaving the vacuoles in their physiological surrounding, i.e. protected by the rigid cell wall. The permeabilisation was monitored by the latency of predominantly cytosolic amino acids and the ATP status of the cells. Functional intactness of the vacuoles within the permeabilised hyphae was demonstrated by maintenance of the pH gradient across the vacuolar membrane as detected by accumulation of the fluorescent dye, Acridine orange. These two methods are well‐suited tools for the in situ assay of intracellular compartmentation of metabolites, for vacuolar transmembrane fluxes in Ashbya gossypii , as well as for the direct access to vacuolar membranes and enzymes of this fungus.