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Plant and Cell Physiology, 2002, Vol. 43, No. 2 143-151
© 2002 Oxford University Press

Activation of Arabidopsis Vacuolar Processing Enzyme by Self-Catalytic Removal of an Auto-Inhibitory Domain of the C-Terminal Propeptide

Miwa Kuroyanagi1, Mikio Nishimura2 and Ikuko Hara-Nishimura1,3

1 Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606–8502 Japan 2 Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444–8585 Japan

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. To clarify the mechanism of maturation and activation of VPE, we expressed the precursors of Arabidopsis {gamma}VPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE) and an unglycosylated preproprotein precursor (ppVPE) which might be unfolded. The N-terminal sequence of pVPE revealed that ppVPE had a 22-amino-acid signal peptide to be removed co-translationally. Under acidic conditions, the 56-kDa pVPE was self-catalytically converted to a 43-kDa intermediate form (iVPE) and then to the 40-kDa mature form (mVPE). N-terminal sequencing of iVPE and mVPE showed that sequential removal of the C-terminal propeptide and N-terminal propeptide produced mVPE. Both iVPE and mVPE exhibited the activity, while pVPE exhibited no activity. These results imply that the removal of the C-terminal propeptide is essential for activating the enzyme. Further removal of the N-terminal propeptide from iVPE is not required to activate the enzyme. To demonstrate that the C-terminal propeptide functions as an inhibitor of VPE, we expressed the C-terminal propeptide and produced specific antibodies against it. We found that the C-terminal propeptide reduced the activity of VPE and that this inhibitory activity was suppressed by specific antibodies against it. Our findings suggest that the C-terminal propeptide functions as an auto-inhibitory domain that masks the catalytic site. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme.

3 Corresponding author: E-mail, ihnishi@gr.bot.kyoto-u.ac.jp; Fax, +81-75-753-4142.


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