Assay for AtSBT1.1 activity in vitro AtSBT1.1-myc was affinity-purified from transgenic Arabidopsis plants as follows: 500 g of seedlings were ground in liquid nitrogen and suspended in 25 mM Tris/HCl pH 7.2, 150 mM NaCl, 0.1% Nonidet P-40 (Calbiochem, http://www.emdbiosciences.com) and 10% glycerol. Anti-c-myc agarose conjugate (200 ll; Sigma) was added to the filtered lysate and incubated for 2 h at 4°C with continuous rotation. The agarose beads with bound AtSBT1.1-myc were recovered by centrifugation at 1000 g for 3 min at 4°C. The beads were washed three times with washing buffer (25 mM Tris/HCl pH 7.2, 150 mM NaCl) and suspended in 25 mM MES/sodium acetate buffer pH 6.0. The reactions were carried out at 32°C in the same buffer supplemented with 2.5 mM CaCl 2 . Parallel purification was performed using transgenic plants transformed with a mutated form (S552A) of AtSBT1.1-myc and the empty vector to obtain material for control reactions. For fluorogenic peptide assays, 40 ll of bead-bound AtSBT1.1-myc were added to a solution containing a final concentration of 50 lM fluorogenic peptide in a buffer consisting of 25 mM MES/ sodium acetate pH 6.0 supplemented with 2.5 mM CaCl 2 . Kinetic assays were performed at 32°C and monitored as fluorescence emission at 420 nm (10 nm slit) following excitation by 320 nm (10 nm slit) in a BioTek spectrophotometer (http://www.biotek. com). The reaction was carried out in 96-well plates (Nunc, http:// www.nuncbrand.com). Control reactions were performed using the same fluorogenic peptide with bead-bound mutated AtSBT1.1 (S552A) and bead-bound myc vector only. AtSBT1.1 incubated with 1 mM PMSF, peptide and reaction buffer, and buffer alone incubated in separate wells served as additional controls. To determine the pH optimum of the reaction, a tri-component buffer system of constant ionic strength was used (Ellis and Morrison, 1982) . This buffer comprised 25 mM acetic acid, 25 mM MES, 50 mM Tris/HCl and 2.5 mM CaCl 2 .