Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles

Abstract

The yeast vacuole is functionally analogous to the mammalian lysosome. Both play important roles in fundamental cellular processes such as protein degradation, detoxification, osmoregulation, autophagy and apoptosis which, when deregulated in humans, can lead to several diseases. Some of these vacuolar roles are difficult to study in a cellular context, and therefore the use of a cell-free system is an important approach to gain further insight into the different molecular mechanisms required for vacuolar function. In the present study, the potentialities of flow cytometry for the structural and functional characterization of isolated yeast vacuoles were explored. The isolation protocol resulted in a yeast vacuolar fraction with a degree of purity suitable for cytometric analysis. Moreover, isolated vacuoles were structurally and functionally intact and able to generate and maintain electrochemical gradients of ions across the vacuolar membrane, as assessed by flow cytometry. Proton and calcium gradients were dissipated by NH 4 Cl and calcimycin, respectively. These results established flow cytometry as a powerful technique for the characterization of isolated vacuoles. The protocols developed in this study can also be used to enhance our understanding of several molecular mechanisms underlying the development of lysosome-related diseases, as well as provide tools to screen for new drugs that can modulate these processes, which have promising clinical relevance.

Identification of plant vacuolar transporters mediating phosphate storage

“…33 P transport assay was performed with isolated yeast vacuoles according to a modified protocol 62 . After incubation with 33 Pi, vacuoles were floated and purified with silicon oil 63 followed by radioactivity measurement. When applied, a final concentration of 4 mM ATP or 10 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added in the uptake solution.…”

Lactoferrin selectively triggers apoptosis in highly metastatic breast cancer cells through inhibition of plasmalemmal V-H+-ATPase

“…Proton pumping activity of V-H + -ATPase in isolated lysosomes/crude membranes was evaluated by measuring ACMA fluorescence quenching in a spectrofluorimeter (LS-5B, Perkin-Elmer) [ 44 ]. The excitation/emission wavelengths were set to 415 nm and 485 nm, respectively.…”

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae

“…Vacuole isolation was performed as previously described [32,33]. After grown until an OD640 = 0.7, 1 l of BY4741 cell suspension was collected, washed twice with ice-cold distilled water, resuspended in washing buffer [5% (w/v) glucose, 10 mM MES (morpholineethanesulfonic acid)-Tris, pH 6.5] and incubated in a shaker at 30°C for 30 min.…”