Poster: Organelle Biogenesis

Abs # 1156: Activation of vacuolar processing enzyme of Arabidopsis by auto-catalytic removal of the C-terminal propeptide as an auto-inhibitory domain

Presenter:	Kuroyanagi, Miwa , kuroyanagi@gr.bot.kyoto-u.ac.jp
Authors	Kuroyanagi, Miwa  (A)   Nishimura, Mikio  (A)   Hara-Nishimura, Ikuko  (B)
Affiliations:	(A): National Institute for Basic Biology (B): Kyoto University

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. Vegetative VPEs have been shown to be up-regulated in lytic vacuoles during senescence and under various stress conditions. To clarify a VPE-mediated processing system in the lytic vacuoles, it is necessary to explore the detailed mechanism of the maturation and activation of vegetative VPE. We expressed the precursors of Arabidopsis gVPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE). 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. The C-terminal propeptide reduced the activity of VPE and the inhibitory activity was suppressed by specific antibodies against it. We show that the C-terminal propeptide of VPE functions as an auto-inhibitory domain of the enzyme. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme. It is possible that VPE-mediated processing system regulate maturation and activation of lytic enzymes that function in programmed cell death of higher plants.