Poster: Protein Targeting & Vesicular Trafficking

Abs # 1185: Ectopic expression of an ER-localized form of PV72, a seed-specific vacuolar sorting receptor, in Arabidopsis leaves

Presenter:	Koumoto, Yasuko , komoto@gr.bot.kyoto-u.ac.jp
Authors	Koumoto, Yasuko  (A)   Watanabe, Etsuko  (A)   Shimada, Tomoo  (A)   Tamura, Kentaro  (A)   Matsushima, Ryo  (A)   Nishimura, Mikio  (B)   Hara-Nishimura, Ikuko  (A)
Affiliations:	(A): Graduate School of Science, Kyoto University (B): Department of Cell Biology, National Institute for Basic Biology

To elucidate the receptor-dependent transport of vacuolar proteins in plant cells, we produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumenal domain of PV72 and an ER-retention signal, HDEL. The expression of PV72-HDEL induced the accumulation of a precursor of a cysteine proteinase, AtALEU, which contains a typical vacuolar targeting signal, NPIR, in the propeptide. The accumulation level of the AtALEU precursor was dependent on that of PV72-HDEL. In contrast, it did not induce the accumulation of a precursor of another cysteine proteinase, RD21, which contains no NPIR. Detailed subcellular localization revealed that both the AtALEU precursor and PV72-HDEL accumulated in the ER fraction. These results suggested that the AtALEU precursor is trapped by PV72-HDEL in the ER and is not transported to the vacuoles. As the AtALEU precursor molecules were co-purified with PV72-HDEL, it was confirmed that they formed a complex. Analysis by surface plasmon resonance showed that the lumenal domain of PV72 bound to the NPIR-containing peptide of the AtALEU precursor in the presence of Ca²⁺ ( K_D = 0.1 mM). The overall results support the hypothesis that an Arabidopsis homolog of PV72 functions as a sorting receptor for the NPIR-containing proteinase. Pumpkin PV72 is a seed-specific receptor that is predicted to sort seed storage proteins to protein storage vacuoles. Our findings suggest that the vacuolar sorting receptors for the protein storage vacuoles and those for the lytic vacuoles share the similar signal-recognition mechanism. First two authors equally contributed to this study.