Analysis of autophagy activated during changes in carbon source availability in yeast cells

Ryo Iwama,Yoshinori Ohsumi

doi:10.1074/jbc.ra118.005698

Abstract

Autophagy is a conserved intracellular degradation system in eukaryotes. Recent studies have revealed that autophagy can be induced not only by nitrogen starvation but also by many other stimuli. However, questions persist regarding the types of conditions that induce autophagy, as well as the particular kinds of autophagy that are induced under these specific conditions. In experimental studies, abrupt nutrient changes are often used to induce autophagy. In this study, we investigated autophagy induction in batch culture on low-glucose medium, in which growth of yeast (Saccharomyces cerevisiae) cells is clearly reflected exclusively by carbon source state. In this medium, cells pass sequentially through three stages: glucose-utilizing, ethanol-utilizing, and ethanol-depleted phases. Using GFP cleavage assay by immunoblotting methods, fluorescence microscopy, and transmission electron microscopy ultrastructural analysis, we found that bulk autophagy and endoplasmic reticulum-phagy are induced starting at the ethanol-utilizing phase, and bulk autophagy is activated to a greater extent in the ethanol-depleted phase. Furthermore, we found that mitophagy is induced by ethanol depletion. Microautophagy occurred after glucose depletion and involved incorporation of cytosolic components and lipid droplets into the vacuolar lumen. Moreover, we observed that autophagy-deficient cells grow more slowly in the ethanol-utilizing phase and exhibit a delay in growth resumption when they are shifted to fresh medium from the ethanol-depleted phase. Our findings suggest that distinct types of autophagy are induced in yeast cells undergoing gradual changes in carbon source availability.

Highlights

Autophagy is a conserved intracellular degradation system in eukaryotes
The yeast S. cerevisiae exhibits diauxie when cultured in glucose-containing medium
We demonstrated that cleavage of Vph1-GFP, a vacuolar membrane protein thought to be cleaved by microautophagy, occurred in the ethanol-utilizing and ethanol-depleted phases (Fig. 7D) and that this cleavage took place independently of the ATG genes (Fig. 7C)

Summary

To whom correspondence should be addressed

With the vacuolar membrane, leading to release of the inner spherical structure, called the autophagic body, into the vacuolar lumen [1]. The yeast begins to use ethanol to generate ATP in the mitochondria [25] This phenomenon is referred to as the “Crabtree effect” and can be explained by glucose repression, a phenomenon in which the presence of glucose suppresses respiration, the use of alternative carbon sources, and gluconeogenesis [26]. We found that yeast growth in batch culture on synthetic medium with low-glucose is reflected exclusively by carbon source state. Using this medium, we investigated autophagy induction in each phase. Our findings suggest that distinct types of autophagy are induced under gradual changes of carbon source availability in yeast

Results

Discussion

Experimental procedures