Friso Giulia, Giacomelli Lisa, Ytterberg A Jimmy, Peltier Jean-Benoit, Rudella Andrea, Sun Qi, Wijk Klaas J van
Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
Plant Cell. 2004 Feb;16(2):478-99. doi: 10.1105/tpc.017814. Epub 2004 Jan 16.
An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.
通过用盐、去污剂和有机溶剂进行连续提取，随后进行多维蛋白质分离步骤（反相高效液相色谱以及一维和二维电泳凝胶）、不同的酶促和非酶促蛋白质裂解技术、质谱分析和生物信息学，对拟南芥外周和类囊体膜整合蛋白质组进行了广泛分析。总共鉴定出154种蛋白质，其中76种（49%）是α螺旋整合膜蛋白。鉴定出27种功能未知但具有预测叶绿体转运肽的新蛋白质，其中17种（63%）是整合膜蛋白。这些新蛋白质可能在类囊体生物发生中起重要作用，包括两种红素氧还蛋白、一种潜在的金属伴侣蛋白和一种新的DnaJ样蛋白。这些数据与我们对富含腔室蛋白质组的分析相结合。我们在100种已知的类囊体定位光合装置蛋白质中鉴定出83种，包括几种新的旁系同源物以及约20种参与蛋白质插入、组装、折叠或蛋白水解的蛋白质。另外16种蛋白质参与翻译，表明类囊体膜表面是蛋白质合成的重要位点。光合装置的高覆盖率以及对低表达水平的已知疏水蛋白质（如cpSecE、Ohp1和Ohp2）的鉴定表明该分析具有出色的动态分辨率。连续提取过程被证明对验证跨膜预测非常有帮助。我们的数据还与其他实验室的叶绿体亚蛋白质组分析进行了交叉关联。所有数据都存放在一个具有多种搜索功能的新的经过整理的质体蛋白质组数据库（PPDB）中（http://cbsusrv01.tc.cornell.edu/users/ppdb/）。这个PPDB将作为植物群落可扩展的资源。
