Poster: Protein targeting & vesicular trafficking

Abs # 650: Protein aggregates are transported to vacuoles by autophagic mechanism under nutrient starvation

Presenter:	Toyooka, Kiminori , toyooka@psc.riken.go.jp
Authors	Toyooka, Kiminori  (A)   Takeuchi, Masaki  (A)   Shimizu, Masami  (A)   Moriyasu, Yuji  (B)   Fukuda, Hiroo  (A)   Matsuoka, Ken  (A)
Affiliations:	(A): Plant Scinece Center, RIKEN (B): School of Food and Nutritional Sciences, University of Shizuoka

Overexpressed fusion protein of cytochrome B5 and red fluorescent protein (CytB5-RFP) distributed in the log-phase tobacco BY-2 cells as the punctate pattern. However, in some case, some of the RFP fluorescence was detectable in the lumen of the vacuole upon reaching to the stationary phase. Here, we address the transport processes of RFP fluorescence into the inside of vacuoles. Ultrastructural and immunocytochemical analyses of CytB5-RFP expressing cells showed that CytB5-RFP was aggregated as a spherical structure without membrane. To examine what is the trigger of such the relocation of CytB5-RFP aggregates at the stationary-grown cells, we incubated the cells with different medium and found that nutrient starvation caused the relocation of RFP fluorescence into the vacuole. When a cysteine protease inhibitor was present in the starvation medium, the relocation of CytB5-RFP was blocked, and the aggregates were found to be within autolysosome-like structures. To examine the involvement of autophagy for the relocation of RFP fluorescence into vacuole, we expressed the fusion protein of yellow fluorescent protein (YFP) and Atg8 homolog of BY-2 (NtAtg8) in the CytB5-RFP expressing cells. The YFP-fluorescence was detectable in the cytoplasm with a few dots. The YFP dots were increased under starvation conditions, and some of them were transported to the inside of large vacuoles. These results indicate the YFP spots are autophagosomes. Furthermore, these autophagosomes of YFP-NtAtg8 appeared to be colocalized with some CytB5-RFP aggregates. These findings suggest that BY-2 cells upon starvation use the autophagic process for degradation of the protein aggregates.