Purification of a proaleurain maturation protease

Aleurain is a cysteine aminopeptidase expressed ubiquitously in plants that serves as a marker for the lytic vacuole. The protease responsible for converting proaleurain, the inactive zymogen, into active form is not known. Using recombinant proaleurain secreted into the medium of Drosophila S2 cells as substrate, we have purified a protease from cauliflower florets that converts proaleurain into a molecular form indistinguishable in size from barley aleurain. This proaleurain maturation protease is a cysteine protease with C-terminal granulin domain, a plant-specific protease that represents only a small set of members of the plant papain-like cysteine protease family. The purified maturation protease uniquely required activation by partial denaturation in 2% SDS, and was active at pH 4.5 but not 5.0 or 5.5. The closely related Arabidopsis cysteine protease with granulin domain, RD21, is a vacuolar enzyme [K. Yamada, R. Matsushima, M. Nishimura, I. Hara-Nishimura, A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves, Plant Physiol. 127 (2001) 1626-1634]. Our work is the first to tie a member of this vacuolar protease family to a specific substrate, and raises the possibility that the proaleurain maturation protease could regulate proaleurain activation in vivo.