Hagiwara-Komoda Yuka, Sugiyama Tomoya, Yamashita Yui, Onouchi Hitoshi, Naito Satoshi
Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan These authors contributed equally to this work. Present address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan.
Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan These authors contributed equally to this work. Present address: Chugai Pharmaceutical Co., Ltd., API Process Development Department, Tokyo, 115-8543 Japan.
Plant Cell Physiol. 2014 Oct;55(10):1779-92. doi: 10.1093/pcp/pcu110. Epub 2014 Aug 20.
Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine γ-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1:GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1:GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.
位于核编码叶绿体蛋白N端区域的叶绿体转运肽序列（cTPs）对其分选至关重要，并且通常在这些蛋白导入叶绿体后从蛋白上被切割下来。拟南芥胱硫醚γ-合酶（CGS）是甲硫氨酸生物合成的首个关键酶，是一种核编码的叶绿体蛋白。拟南芥CGS具有一个对酶活性而言并非必需的N端延伸区域。这个N端延伸区域包含cTP和几个功能域，包括一个MTO1区域，它是编码CGS的CGS1转录后反馈调节的顺式元件。先前的一份报告表明CGS的cTP切割位点位于MTO1区域的上游。然而，蛋白分选所需的区域尚未得到分析。在本研究中，我们通过使用一种嵌合蛋白Ex1:GFP进行功能分析，以阐明叶绿体靶向所需的区域，其中包含N端延伸的CGS1外显子1编码区域用绿色荧光蛋白进行了标记。MTO1区域上游的序列负责高效的叶绿体靶向以及避免错误分选至线粒体。我们的数据还表明Ex1:GFP的主要N端是Ala91，它位于MTO1区域的紧邻下游，并且MTO1区域并不保留在叶绿体中积累的成熟Ex1:GFP中。这些发现表明N端可切割的前序列在蛋白分选和调节基因表达方面具有双重功能。我们的研究突出了拟南芥CGS cTP在叶绿体靶向蛋白中的独特特性。
