Assessing autophagic flux in yeast.

Abstract

Autophagy is an intracellular recycling program that is ubiquitously present in eukaryotes, where it is crucial for the maintenance of cellular homeostasis. Thereby, superfluous or damaged proteins, organelles and cytoplasmic portions are targeted either in bulk or selectively for delivery to the lysosome (or vacuole in yeast). There, they are subsequently digested to provide substrates and building blocks. The core components as well as the regulatory circuits governing this well-orchestrated catabolic pathway are highly conserved among eukaryotes. The baker's yeast Saccharomyces cerevisiae has contributed fundamentally to the understanding of the molecular principles and the discovery of major regulators of autophagy, and remains a favorable model organism for the study of different aspects of this pathway. Here, we provide detailed protocols for two of the main established assays used to monitor autophagic activity in yeast, the green fluorescent protein (GFP) liberation assay and the alkaline phosphatase (ALP) activity assay. Notably, both assays follow the delivery of autophagosomal cargo into the yeast vacuole and thus provide a valuable tool for the measurement of actual autophagic flux. We further recapitulate experimental setups for both assays and provide information on how they can be used to study non-selective and selective forms of autophagy in yeast.