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首页> 外文期刊>Biochemistry >Mutagenesis of some positive and negative residues occurring in repeat triad residues in the ADP/ATP carrier from yeast
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Mutagenesis of some positive and negative residues occurring in repeat triad residues in the ADP/ATP carrier from yeast

机译：酵母ADP / ATP载体中重复三联残基中出现的一些正负残基的诱变

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In AAC2 from Saccharomyces cerevisiae, nine additional charged residues (six positive, three negative) were neutralized by mutagenesis following the previous mutation of six arginines. Oxidative phosphorylation (OxPhos) in cells and mitochondria, the expression level of AAC protein, and the various transport modes of AAC in the reconstituted system were measured. Mutations are: within the first helix at K38A which is exclusive for AAC; K48A, and R152A, part of a positive triad occurring in the matrix portion of each repeat; two matrix lysines, K179M and K182I, and the negative triad helix-terminating residues, E45G, D149S, D249S. Cellular ATP synthesis (OxPhos) is nearly completely inhibited in K48A, R152A, D149S, and D249S, but still amounts to 10% in K38A and between 30% and 90% in the gly+ mutants K179M, K179I + K182I, and E45G. Comparison of the AAC content measured by ELISA and the binding of [3H]CAT and [3H]BKA reveals discrepancies in K48A, D149S, and D249S mitochondria, which provide evidence that these mutations largely abolish inhibitor binding. Also these mitochondria have undetectable OxPhos. Differently in K38A, CAT and BKA binding are retained at high AAC levels but OxPhos is very low. This reveals a special functional role of K38, different from the more structural role of R152, K48, D149, and D249. Transport activity was measured with reconstituted AAC. The electroneutral ADP/ADP exchange of gly- mutants is largely or fully suppressed in K48A, D149S, and D249S. K38A and R152A are still active at 18% and 30% of wt. The other three exchange modes, ATP/ADP, ADP/ATP, and ATP/ATP, are nearly suppressed in all gly- mutants but remain high in gly+ mutants. ATP-linked modes are higher than the ADP/ADP mode in gly+ but lower in gly- mutants, resulting in an exchange mode inversion (EMI). In the competition for AAC2 transport capacity, the weak ATP exporting modes are suppressed by the much stronger unproductive ADP/ADP mode causing inhibition of OxPhos. Together with previous results all members of three charge triads are now mutagenized, revealing drastic functional rotatory asymmetries within the three repeat domains. In the intrahelical arginine triad the third (R294A), in the positive matrix triad the second (R152A), and in the helix-terminating negative triad the first (E45G) still show high activity.
机译：在来自酿酒酵母的AAC2中，先前突变了六个精氨酸后，通过诱变中和了另外九个带电残基（六个阳性，三个阴性）。测量了细胞和线粒体中的氧化磷酸化（OxPhos），AAC蛋白的表达水平以及AAC在重组系统中的各种运输方式。突变是：在A38专用的K38A的第一个螺旋内; K48A和R152A，出现在每个重复序列的矩阵部分中的三元组的一部分;两个基质赖氨酸K179M和K182I，以及负三联体螺旋终止残基E45G，D149S，D249S。细胞ATP合成（OxPhos）在K48A，R152A，D149S和D249S中几乎完全被抑制，但在K38A中仍达到10％，在gly +突变体K179M，K179I + K182I和E45G中达到30％至90％。通过ELISA测定的AAC含量与[3H] CAT和[3H] BKA结合的比较表明，K48A，D149S和D249S线粒体存在差异，这提供了这些突变在很大程度上消除了抑制剂结合的证据。这些线粒体还具有不可检测的OxPhos。与K38A不同的是，CAT和BKA结合在高AAC水平下得以保留，但OxPhos却非常低。这揭示了K38的特殊功能作用，不同于R152，K48，D149和D249的更多结构作用。运输活性用重构的AAC测量。在K48A，D149S和D249S中，gly突变体的电中性ADP / ADP交换被大大或完全抑制。 K38A和R152A仍具有18％和30％wt的活性。其他三种交换模式，ATP / ADP，ADP / ATP和ATP / ATP，在所有gly-突变体中都几乎被抑制，但在gly +突变体中仍然很高。 ATP连锁模式在gly +中高于ADP / ADP模式，但在gly-突变体中更低，导致交换模式倒置（EMI）。在竞争AAC2的运输能力中，弱得多的ATP出口模式被强得多的非生产性ADP / ADP模式所抑制，从而抑制了OxPhos。连同先前的结果，三个电荷三联体的所有成员现在都被诱变，从而揭示了三个重复域内剧烈的功能性旋转不对称。在螺旋形精氨酸三联体中，第三个（R294A），在正基质三联体中，第二个（R152A），在螺旋终止的三联体中，第一个（E45G）仍然显示高活性。

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