Bohrer Anne-Sophie, Yoshimoto Naoko, Sekiguchi Ai, Rykulski Nicholas, Saito Kazuki, Takahashi Hideki
Department of Biochemistry and Molecular Biology, Michigan State University East Lansing, MI, USA.
Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
Front Plant Sci. 2015 Jan 5;5:750. doi: 10.3389/fpls.2014.00750. eCollection 2014.
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. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3, and -4) 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 Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUG(Met1) to produce plastid-targeted ATPS2 pre-proteins or at AUG(Met52) or AUG(Met58) within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUG(Met52) or AUG(Met58) was verified by expressing a tandem-fused synthetic gene, ATPS2 (5'UTR-His12) :Renilla luciferase:ATPS2 (Ile13-Val77) :firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUG(Met52) and AUG(Met58) significantly reduced the firefly luciferase activity, while AUG(Met52) was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUG(Met52) or AUG(Met58) 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 Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.
植物将无机硫酸盐同化为含硫的重要代谢产物。ATP硫酸化酶（ATPS）是催化植物质体和细胞质中硫酸盐同化途径关键起始步骤的酶。拟南芥有四个ATPS基因（ATPS1、-2、-3和-4），它们编码的ATPS前体蛋白含有用于质体靶向的N端转运肽序列，然而，细胞质中ATPS的基因身份尚未得到验证。在这里，我们表明拟南芥ATPS2双重编码质体和细胞质中的ATPS同工型，通过在AUG（Met1）起始翻译以产生靶向质体的ATPS2前体蛋白，或在转运肽内的AUG（Met52）或AUG（Met58）起始翻译以使ATPS2留在细胞质中来区分它们的亚细胞定位。通过在拟南芥原生质体中单个组成型活性CaMV 35S启动子下表达串联融合的合成基因ATPS2（5'UTR-His12）:海肾荧光素酶:ATPS2（Ile13-Val77）:萤火虫荧光素酶，并检测与ATPS2的N端分裂部分框内翻译的两种不同荧光素酶的活性，验证了ATPS2在AUG（Met52）或AUG（Met58）处的翻译起始。在AUG（Met52）和AUG（Met58）处引入错义突变显著降低了萤火虫荧光素酶活性，而AUG（Met52）是替代翻译起始的相对优选位点。从AUG（Met52）或AUG（Met58）开始的荧光素酶融合蛋白的活性不受硫酸盐条件变化的调节。ATPS2-GFP融合蛋白在拟南芥原生质体和转基因系中的表达进一步证明了ATPS2在质体和细胞质中的双重定位，同时它们也受主要在叶表皮细胞、保卫细胞、维管组织和根中发现的组织特异性ATPS2启动子活性的控制。
