烟草BY-2细胞在无蔗糖培养基中进行自噬,这是在这些条件下主要负责细胞内蛋白质降解的过程。空泡H()-ATPase抑制剂concanamycinA和bafilomycinA1抑制自噬,导致自噬体在中央液泡中积累。这种积累在自噬抑制剂3-甲基腺嘌呤的存在下不会发生,在存在半胱氨酸蛋白酶抑制剂E-64c的情况下,可卡霉素又抑制了自溶酶体的积累。电子显微镜显示,自噬体不仅积累在中央液泡中,而且,在用可卡霉素治疗的细胞质中更频繁地观察到自噬体样结构,表明可卡霉素除了提高中央液泡的pH值外,还影响自噬体的形态。使用组成型表达自噬小体标记蛋白Atg8和绿色荧光蛋白(GFP)的融合蛋白的BY-2细胞,我们通过荧光显微镜观察了自噬体的出现,这是自噬的可靠形态标记,以及将融合蛋白加工成GFP,它是自噬的生化标志物。一起,这些结果表明,在BY-2细胞的自噬过程中,空泡型H()-ATPase参与了自噬体到自溶酶体的成熟步骤。自噬体在中央液泡中的积累是自噬发生的标志;然而,这并不一定意味着中央液泡是细胞质降解的部位。
Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors
concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and
concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with
concanamycin, suggesting that
concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by
concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.