3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions.

Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.