Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization.

Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.