Poster: Protein Targeting & Vesicular Trafficking

Abs # 1174: Light-induced degradation of green fluorescent protein in the vacuoles of Arabidopsis

Presenter:	Tamura, Kentaro , tamura@gr.bot.kyoto-u.ac.jp
Authors	Tamura, Kentaro  (A)   Shimada, Tomoo  (A)   Ono, Eiichiro  (B)   Tanaka, Yoshikazu  (B)   Nagatani, Akira  (A)   Hara-Nishimura, Ikuko  (A)
Affiliations:	(A): Department of Botany, Graduate School of Science, Kyoto University (B): Institute for Advanced Technology, Suntory Ltd

Green fluorescent protein (GFP) from luminescent jellyfish Aequorea victoria has been widely used as a reporter in the determination of protein localization the visualization of organelles in living plant cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions and that the fluorescence rapidly disappeared under light conditions. Subcellular fractionation of the transgenic plants showed that the 30-kDa GFP fusion protein was trimmed to produce a smaller 27-kDa GFP in the vacuoles. The 27-kDa vacuolar GFP was rapidly degraded within 1 h in the light. An inhibitor of vacuolar type H⁺-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. This implies that papain-type proteinase(s) with an acidic optimum pH are involved in GFP degradation. Cycloheximide had no effect on GFP degradation, indicating that de novo synthesis of such proteinase(s) is not necessary for GFP degradation. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. GFP degradation in the vacuoles was induced by blue light. The overall results suggest that blue light induced a conformational change in GFP and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs.