Porras Pablo, McDonagh Brian, Pedrajas Jose Rafael, Bárcena J Antonio, Padilla C Alicia
Max Delbrueck Center, D-13125 Berlin-Buch, Germany.
Biochim Biophys Acta. 2010 Apr;1804(4):839-45. doi: 10.1016/j.bbapap.2009.12.012. Epub 2009 Dec 28.
We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.
我们之前已经表明，酿酒酵母中的谷氧还蛋白2（Grx2）定位于3个不同的亚细胞区室，即细胞质、线粒体基质和外膜，这是一个单一基因不同翻译后加工的结果。在确定了导致这一显著现象的机制后，我们现在旨在确定这种亚细胞定位的多样性是否与Grx2同工型的结构和功能差异相关。我们通过质谱法确定了可溶性线粒体基质Grx2的N端序列，并通过线粒体加工肽酶（MPP）确定了确切的切割位点。由于这种切割，与细胞质形式相比，线粒体基质Grx2同工型在N端具有一个碱性四肽延伸。与这种结构差异的功能关系是，线粒体Grx2在催化线粒体二硫醇二氢硫辛酰胺还原GSSG方面表现出明显更高的活性。我们制备了在前序列内关键残基上发生突变的Grx2突变体，以将蛋白质导向一个单一的细胞区室，即细胞质、线粒体膜或基质，并分析了它们的功能表型。仅在细胞质中表达Grx2的菌株与缺乏该基因的菌株对H(2)O(2)的敏感性相同，而仅在线粒体基质中表达Grx2的菌株更具抗性。关键碱性残基的突变极大地影响了蛋白质的细胞命运，表明Grx2结构和功能特性的进化多样化严格依赖于靶向信号肽的序列。
