Edvardsson Anna, Shapiguzov Alexey, Petersson Ulrika A, Schröder Wolfgang P, Vener Alexander V
Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden.
Biochemistry. 2007 Aug 21;46(33):9432-42. doi: 10.1021/bi700426q. Epub 2007 Jul 27.
The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.
亲免素的生理作用尚不清楚，但许多亲免素具有肽基脯氨酰异构酶（PPIase）活性，并且在迄今为止检测的所有生物体中均已发现它们，这意味着它们参与了基本的蛋白质折叠过程。高等植物拟南芥的叶绿体类囊体腔中含有多达16种亲免素（5种亲环素和11种FKBP），但其中只有两种，即AtCYP20 - 2和AtFKBP13，被发现是有活性的PPIase，这表明该细胞区室中的其他亲免素可能已经丧失了其假定的PPIase活性。为了评估这种可能性，我们在酶学和定量蛋白质组学分析中对两个独立的缺失AtCYP20 - 2的拟南芥敲除系进行了表征。两个突变体类囊体腔制剂中的PPIase活性与相应野生型制剂的活性相当，并且对突变体和野生型的腔蛋白进行的比较二维差异凝胶电泳分析表明，在AtCYP20 - 2缺陷型植物中，没有一种潜在的PPIase含量更高。酶学分析确定，突变体类囊体腔中的所有PPIase活性均归因于AtFKBP13，并且该酶的氧化激活补偿了AtCYP20 - 2的缺失。因此，对腔亲环素和FKBP的潜在催化结构域的序列分析表明，只有AtCYP20 - 2和AtFKBP13具有在早期对人亲环素A和FKBP12的研究中发现的对PPIase活性至关重要的所有氨基酸残基。因此，除AtCYP20 - 2和AtFKBP13外，拟南芥叶绿体类囊体腔中的亲免素似乎均不具有针对肽底物的脯氨酰异构酶活性。
