Halls, C.E.3; Rogers, S. W.3; Ouffattole, M.3; Østergaard, O.3; Svensson, Birte4; Rogers, J. C.3
1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark3 unknown4 Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark
A Kunitz-type protease inhibitor co-purified from cauliflower florets with a granulin domain cysteine protease that cleaved barley proaleurain to yield a molecular form the same size as that for mature aleurain. The purified cauliflower protease required treatment with SDS detergent to become active. This observation raised the question of whether the protease inhibitor might have the ability to interact with the granulin domain protease. Here we express an Arabidopsis homolog of the protease inhibitor as a recombinant protein and demonstrate that it is a potent inhibitor of the recombinant proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts, it colocalized with BP-80, a vacuolar sorting receptor that interacts with proaleurain and traffics to prevacuolar compartments for lytic vacuoles. Our results indicate that the cauliflower and Arabidopsis protease inhibitors would traffic through cellular compartments where proaleurain also traffics. Their ability to inhibit a cysteine protease implicated in maturation of proaleurain to active form at the acidic pH found in vacuoles raises the possibility that they could participate in regulating activation of aleurain. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
Plant Science, 2006, Vol 170, Issue 6, p. 1102-1110