*4.4. Protein Expression, Purification, and ATPase Assay*

The *OsAAA-ATPase1* sequence was amplified by PCR and cloned into the sites between BglI and HidIIIp of pET32a (Novagen) as a His-tag fusion protein; then, that was transfected into the *Escherichia coli* Origami strain BL21(Lys). The set of primers used was as follows: OsAAA1BglII 5- -GTAGATCTCTTGAGACAAATGGAGGCGACG-3- ; OsAAA1HindIII 5- -GCTAAGCTTCTACTTATCCTTCCCGACCAC-3- . Expression of the protein was induced for 4 h at 25 ◦C with 0.5 mmol/L isopropyl β-d-1-thiogalactopyranoside. *Escherichia coli* cells were pelleted by centrifugation, resuspended in lysis buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 10 mmol/L imidazole), and sonicated. After the cell debris was removed by centrifugation (12,000 × g, 10 min, 4 ◦C), the supernatant was loaded onto a High Affinity Ni-Charged Resin (GE Healthcare, Buckinghamshire, UK), washed with washing buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 10 mmol/L imidazole), and eluted with elution buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 180 mmol/L imidazole). ATPase activity was measured by the malachite green-based colorimetric method using the QuantiChromTM ATPase/GTPase activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). The elution buffer was used as the negative control, and an ATPase from potatoes (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control. One unit is defined as the amount of enzyme that catalyzes the production of 1 μM of free phosphate per minute under the assay conditions.

#### *4.5. Subcellular Localization*

For subcellular localization of OsAAA-ATPase1, the plasmid pSAT6-AFP-C1-OsAAA1 was transformed into protoplasts prepared from etiolated seedlings as previously described [52]. As a control for cytoplasmic localization, the pSAT-mCherry construct was co-transformed. Fluorescence was examined under a confocal microscope (Leica Microsystems, Wetzlar, Germany) 16 h after transformation.
