Interdisciplinary Plant Group and Department of Biochemistry, University of Missouri, Christopher S Bond Life Sciences Center, Columbia, Missouri 65211, United States.
J Proteome Res. 2011 Sep 2;10(9):4076-87. doi: 10.1021/pr200263m. Epub 2011 Aug 3.
The 14-3-3-protein family is prominently expressed during seed filling and modulates protein interactions and enzymatic activities, in a phosphorylation-dependent manner. To investigate the role(s) of 14-3-3 proteins in oilseed development, we have begun to characterize the Arabidopsis thaliana 14-3-3 "interactome" for two phylogenetically distinct isoforms. Proteins from developing Arabidopsis seed were incubated with a Sepharose affinity matrix containing covalently bound recombinant Arabidopsis 14-3-3 isoforms chi (χ) or epsilon (ε). Eluted proteins were quantitatively identified using GeLC-MS/MS coupled to spectral counting. Analysis of nine biological replicates revealed a total of 104 putative 14-3-3 binding proteins eluted from this affinity matrix compared to controls. Interestingly, these results imply that χ and ε could have distinct preferences for client proteins. Both isoforms interact with client proteins involved in various metabolic pathways, including glycolysis and de novo fatty acid synthesis. These results suggest 14-3-3 proteins interact with multiple biochemical processes of Arabidopsis seed development. Furthermore, these data suggest isoform specificity of client proteins and possibly even functional specialization between the 14-3-3 isoforms χ and ε in Arabidopsis seed development.
14-3-3 蛋白家族在种子灌浆过程中表达明显，以磷酸化依赖的方式调节蛋白质相互作用和酶活性。为了研究 14-3-3 蛋白在油料种子发育中的作用，我们开始对两种系统发育上不同的拟南芥 14-3-3“相互作用组”的两个同工型进行特征描述。用含有共价结合的重组拟南芥 14-3-3 同工型 chi（χ）或 epsilon（ε）的琼脂糖亲和基质孵育发育中的拟南芥种子蛋白质。使用 GeLC-MS/MS 结合光谱计数定量鉴定洗脱的蛋白质。对九个生物学重复的分析显示，与对照相比，从这种亲和基质中总共洗脱了 104 种可能的 14-3-3 结合蛋白。有趣的是，这些结果表明 χ 和 ε 可能对客户蛋白有不同的偏好。这两种同工型都与参与各种代谢途径的客户蛋白相互作用，包括糖酵解和从头脂肪酸合成。这些结果表明 14-3-3 蛋白与拟南芥种子发育的多个生化过程相互作用。此外，这些数据表明客户蛋白的同工型特异性，甚至在拟南芥种子发育中 χ 和 ε 同工型之间存在功能专业化。
