A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast S. cerevisiae

Ravi Manjithaya,Shashank Rai,Piyush Mishra

doi:10.1016/j.bbrep.2017.07.008

Abstract

BackgroundMacroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions. ObjectiveIn search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast S. cerevisiae that could measure both selective and general autophagy in real time. MethodsFirefly and Renilla luciferase reporter genes were cloned under POT-1 promoter. Using fatty acid medium the promoter was induced and the luciferase cargo was allowed to build up. The cells were then transferred to starvation conditions to stimulate autophagy and the degradation of luciferase markers was followed with time. Results and conclusionThe assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.

Highlights

Autophagy, a process of cellular self-cannibalization, is an essential recycling mechanism in eukaryotes
Development of dual luciferase assay for measuring autophagic flux
The principle of the assay involves simultaneously building up the levels of firefly luciferase and Renilla luciferase during peroxisome biogenesis and following the degradation of the luciferase activities over time, upon induction of autophagy

Summary

Introduction

A process of cellular self-cannibalization, is an essential recycling mechanism in eukaryotes This evolutionarily conserved process involves selective or non-selective degradation of damaged and redundant cellular components by enveloping them inside double membranous structures called autophagosomes and escorting them to the degradation compartments like vacuole (in yeast) or lysosomes (in mammalian cells) The degradation products such as amino acids are recycled back to cytoplasm and utilized in various metabolic pathways [1,2,3,4]. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy

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Conclusion