AUTHOR=Bohrer Anne-Sophie , Yoshimoto Naoko , Sekiguchi Ai , Rykulski Nicholas , Saito Kazuki , Takahashi Hideki 

TITLE=Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana

JOURNAL=Frontiers in Plant Science

VOLUME=5

YEAR=2015

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2014.00750

DOI=10.3389/fpls.2014.00750

ISSN=1664-462X

ABSTRACT=<p>Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS) is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. <italic>Arabidopsis thaliana</italic> has four <italic>ATPS</italic> genes (<italic>ATPS1, –2, –3</italic>, and <italic>–4</italic>) encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that <italic>Arabidopsis ATPS2</italic> dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUG<sup>Met1</sup> to produce plastid-targeted ATPS2 pre-proteins or at AUG<sup>Met52</sup> or AUG<sup>Met58</sup> within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUG<sup>Met52</sup> or AUG<sup>Met58</sup> was verified by expressing a tandem-fused synthetic gene, <italic>ATPS2</italic><sub>(5′<italic>UTR-His12</italic>)</sub><italic>:Renilla luciferase:ATPS2</italic><sub>(<italic>Ile</italic>13−<italic>Val</italic>77)</sub><italic>:firefly luciferase</italic>, under a single constitutively active CaMV 35S promoter in <italic>Arabidopsis</italic> protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUG<sup>Met52</sup> and AUG<sup>Met58</sup> significantly reduced the firefly luciferase activity, while AUG<sup>Met52</sup> was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUG<sup>Met52</sup> or AUG<sup>Met58</sup> was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in <italic>Arabidopsis</italic> protoplasts and transgenic lines, while they were also under control of tissue-specific <italic>ATPS2</italic> promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.</p>