High throughput analysis of vacuolar acidification.

Autor: Zhang C; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA., Balutowski A; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA., Feng Y; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA., Calderin JD; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA., Fratti RA; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA; Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA. Electronic address: rfratti@illinois.edu.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2022 Dec 01; Vol. 658, pp. 114927. Date of Electronic Publication: 2022 Sep 24.
DOI: 10.1016/j.ab.2022.114927
Abstrakt: Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is lumenal pH. In the endocytic and secretory pathways, lumenal pH is controlled by isoforms and concentration of the vacuolar-type H + -ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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