Poster: Protein Targeting & Vesicular Trafficking

Abs # 1186: Relationship between the degradation of RFP-aggregates and autophagic mechanism in Tobacco BY-2 cells

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

We have been analyzing the vacuolar targeting mechanism in plant cells using the BY-2 cells as a model. We found that the 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. Ultrastructual analysis of CytB5-RFP expressing cells showed that CytB5-RFP was aggregated in the lipid body-like structures. However, in some case, some of the RFP fluorescence was detectable in the lumen of the vacuole upon reaching to the stationary phase. To address what is the trigger of such the relocation of CytB5-RFP aggregates at the stationary-grown cells, we incubated the cells at log-phase in media with the different composition. Hormone-free medium did not cause the redistribution of the fluorescence. In contrast, sucrose-free or nitrogen-free medium caused the relocation of RFP fluorescence into the vacuole. These results indicated that the degradation of the aggregates was induced by starvation condition. 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 membrane-bound structures. To examine the involvement of autophagy for the relocation of RFP fluorescence into vacuole, we expressed the fusion protein of yellow fluorescent protein and Apg8 homolog of BY-2 in the CytB5-RFP expressing cells. The YFP-fluorescence was detactable in the cytoplasm with few punctate spots. The punctate spots increased under starvation conditions, and some of them appeared to be colocalized with some CytoB5-RFP aggregates. These findings suggest that BY-2 cells upon starvation may use the autophagic process for degradation of the aggregates.