Georg-August-University of Goettingen, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany.
J Exp Bot. 2010 Mar;61(5):1441-53. doi: 10.1093/jxb/erq014. Epub 2010 Feb 11.
Leaf peroxisomes are fragile, low-abundance plant cell organelles that are difficult to isolate from one of the few plant species whose nuclear genome has been sequenced. Leaf peroxisomes were enriched at high purity from spinach (Spinacia oleracea) and approximately 100 protein spots identified from 2-dimensional gels by a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing. In addition to the predominant enzymes involved in photorespiration and detoxification, several minor enzymes were detected, underscoring the high sensitivity of the protein identification. The tryptic peptides of three unknown proteins shared high sequence similarity with Arabidopsis proteins that carry putative peroxisomal targeting signals type 1 or 2 (PTS1/2). The apparent Arabidopsis orthologues are a short-chain alcohol dehydrogenase (SDRa/IBR1, At4g05530, SRL>) and two enoyl-CoA hydratases/isomerases (ECHIa, At4g16210, SKL>; NS/ECHId, At1g60550, RLx(5)HL). The peroxisomal localization of the three proteins was confirmed in vivo by tagging with enhanced yellow fluorescent protein (EYFP), and the targeting signals were identified. The single Arabidopsis isoform of naphthoate synthase (NS) is orthologous to MenB from cyanobacteria, which catalyses an essential reaction in phylloquinone biosynthesis, a pathway previously assumed to be entirely compartmentalized in plastids in higher plants. In an extension of a previous study, the present in vivo targeting data furthermore demonstrate that the enzyme upstream of NS, chloroplastic acyl-CoA activating enzyme isoform 14 (AAE14, SSL>), is dually targeted to both plastids and peroxisomes. This proteomic study, extended by in vivo subcellular localization analyses, indicates a novel function for plant peroxisomes in phylloquinone biosynthesis.
叶绿体过氧化物酶体是一种脆弱的、低丰度的植物细胞细胞器，很难从少数核基因组已测序的植物物种中分离出来。我们从菠菜（Spinacia oleracea）中高纯度地富集了叶绿体过氧化物酶体，并通过液相色谱-串联质谱（LC-MS/MS）和从头测序相结合的方法，从 2 维凝胶中鉴定了大约 100 个蛋白质斑点。除了参与光呼吸和解毒的主要酶外，还检测到几种次要酶，这突出了蛋白质鉴定的高灵敏度。三种未知蛋白质的胰蛋白酶肽与拟南芥具有假定过氧化物酶体靶向信号 1 或 2（PTS1/2）的蛋白质具有高度的序列相似性。明显的拟南芥同源物是一种短链醇脱氢酶（SDRa/IBR1，At4g05530，SRL>）和两种烯酰-CoA 水合酶/异构酶（ECHIa，At4g16210，SKL>；NS/ECHId，At1g60550，RLx(5)HL）。通过用增强型黄色荧光蛋白（EYFP）标记这三种蛋白质，在体内证实了它们的过氧化物酶体定位，并确定了靶向信号。苯甲酸盐合酶（NS）的单个拟南芥同工酶与蓝细菌中的 MenB 同源，后者催化质体醌生物合成中的一个必需反应，该途径以前被认为完全局限于高等植物的质体中。在之前研究的扩展中，本研究的体内靶向数据进一步表明，NS 上游的酶，质体酰基辅酶 A 激活酶同工型 14（AAE14，SSL>），双重靶向质体和过氧化物酶体。这项蛋白质组学研究，通过体内亚细胞定位分析进一步扩展，表明植物过氧化物酶体在质体醌生物合成中具有新的功能。
