From the Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259-S2-12 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.
From the Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259-S2-12 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan
J Biol Chem. 2019 Apr 5;294(14):5590-5603. doi: 10.1074/jbc.RA118.005698. Epub 2019 Feb 12.
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 () 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.
自噬是真核生物中一种保守的细胞内降解系统。最近的研究表明，自噬不仅可以由氮饥饿诱导，还可以由许多其他刺激诱导。然而，关于诱导自噬的条件类型以及在这些特定条件下诱导的特定类型的自噬，仍存在一些问题。在实验研究中，通常使用突然的营养变化来诱导自噬。在这项研究中，我们研究了在低葡萄糖培养基中的分批培养中自噬的诱导，其中酵母（）细胞的生长仅由碳源状态明确反映。在这种培养基中，细胞依次经历三个阶段：葡萄糖利用、乙醇利用和乙醇耗尽阶段。通过 GFP 切割分析免疫印迹方法、荧光显微镜和透射电子显微镜超微结构分析，我们发现自噬和内质网自噬从乙醇利用阶段开始诱导，并且在乙醇耗尽阶段更强烈地激活了自噬。此外，我们发现乙醇耗尽诱导了线粒体自噬。微自噬发生在葡萄糖耗尽后，涉及细胞质成分和脂质滴的掺入到液泡腔中。此外，我们观察到自噬缺陷细胞在乙醇利用阶段生长更慢，并且当它们从乙醇耗尽阶段转移到新鲜培养基时，生长恢复会出现延迟。我们的研究结果表明，在酵母细胞中，逐渐改变碳源可用性会诱导不同类型的自噬。
