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Poster: Protein Targeting & Vesicular Trafficking

Abs # 1189: E64d, an inhibitor of papain family proteases, inhibits endocytosis and degradation of plasma membrane proteins in BY-2 cells and arabidopsis root cells

Presenter: Yamada, Kenji , kyamada@nibb.ac.jp
AuthorsYamada, Kenji  (B) (A)  Nishimura, Mikio  (A)   Hara-Nishimura, Ikuko  (B)  
Affiliations: (A): Department of Cell Biology, National Institute for Basic Bioplgy
(B): Graduate School of Science, Kyoto University

Plasma membrane proteins are transported to vacuoles/lysosomes for degradation in animal and yeast cells. The mechanism of transport and degradation of plasma membrane proteins has not been well characterized. We treated tobacco BY-2 cells with a red fluorescent dye, FM4-64, which is known to be incorporated by endocytosis. The red fluorescence was first detected on plasma membrane and then on vacuolar membrane. Because of the efficient and rapid deliver to the vacuoles, red-fluorescent endosomes were scarcely detectable in BY-2 cells. We found that FM4-64-stainable small vesicles were accumulated surround nucleus in BY-2 cells that had been treated with E64d, an inhibitor of papain family proteases, in sucrose-free condition. This result indicates that E64d inhibits the fusion of endosomes with vacuoles in BY-2 cells under sucrose-free condition. The accumulation of endosomes was also found in roots of Arabidopsis seedlings that had been treated with E64d. To clarify how to degrade plasma membrane proteins, we used two transgenic Arabidopsis plants which constitutively expressed a fusion protein of GFP and a plasma membrane protein (LTI6b or PIP2a-GFP). The GFP fluorescence was observed on plasma membrane of the root cells of these transgenic plants, but not inside of the cells. After treatment of the transgenic plants with E64d, the intensity of the GFP fluorescence on plasma membrane was not changed. Interestingly, treatment with E64d induced the GFP-fluorescent vesicles inside of the root cells. The fluorescence was not detected on the vacuolar membrane of the cells. Taken together, these results suggest that E64d inhibits degradation of plasma membrane proteins in endosomes.

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