Autophagy in Tobacco BY-2 Cells Cultured under Sucrose Starvation Conditions: Isolation of the Autolysosome and its Characterization

Abstract

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.

Ultrastructure of autophagy in plant cells

“…same group, 29 the same vesicles were simply called autolysosomes without further justification. The same mistake was made in other papers by members of this group.…”

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

“…However, there are only limited evidences for this model in plant cells (Rose et al, 2006;Toyooka et al, 2006;Katsiarimpa et al, 2011;Takatsuka et al, 2011;Hanamata et al, 2012). SH3P2-GFP signals were shown to translocate onto the autophagosome membrane upon autophagy induction in Arabidopsis cells (Figures 1B and 2B), indicating its involvement in membrane progression of autophagosome formation.…”

“…Membrane sources, including the endoplasmic reticulum (ER), mitochondria, ER-mitochondria contact sites, ER-Golgi intermediate compartment, Golgi apparatus, and plasma membrane have been postulated to contribute to autophagosome formation in yeast and/or mammalian cells (Axe et al, 2008;Hayashi-Nishino et al, 2010a, 2010bHailey et al, 2010;Noda et al, 2011;Ohashi and Munro, 2010;Ravikumar et al, 2010;Ge et al, 2013;Hamasaki et al, 2013). In plants, however, most observations of autophagosome-related structures have been based on the classical features of autophagosomes: their isolated double-membrane appearance, enclosed autophagic cargos, and labeling with an autophagosome marker (Rose et al, 2006;Toyooka et al, 2006;Katsiarimpa et al, 2011;Takatsuka et al, 2011;Hanamata et al, 2012). So far, only one electron microscopy (EM) study has shown direct labeling of a double-membrane autophagosome structure in Arabidopsis thaliana with antibodies against the autophagosomal marker ATG8 (Reyes et al, 2011).…”

Genes for Plant Autophagy: Functions and Interactions

“…Autolysosomes in tobacco culture cells are yet another structure that participates in autophagic processes (Moriyasu and Ohsumi, 1996;Takatsuka et al, 2011). The autolysosomes increase in number when tobacco cells are cultured without sucrose and treated with a cysteine protease inhibitor.…”

“…Working with starved Arabidopsis suspensioncultured cells, Marty and colleagues also observed a doublemembrane-bound structure (0.6-2.5 µm in diameter; without a protease inhibitor) called an autophagic vacuole (Rose et al, 2006). Interestingly, at least some of the autolysosome membrane appears to come from the plasma membrane (Takatsuka et al, 2011). The relationship of autolysosomes and autophagic vacuoles with similar organelles such as endocytic multivesicular bodies (also called prevacuolar compartments), ATI bodies , and senescence-associated vacuoles (Otegui et al, 2005) may be a subject for future research.…”