Müller V, Heidkämper D, Nelson D R, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, 80336 Munich, Germany.
Biochemistry. 1997 Dec 16;36(50):16008-18. doi: 10.1021/bi971867l.
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.
在酿酒酵母的AAC2中，继先前六个精氨酸发生突变后，又有九个额外的带电残基（六个带正电，三个带负电）通过诱变被中和。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重组系统中AAC的各种转运模式。突变包括：在第一个螺旋内的K38A，这是AAC特有的；K48A和R152A，它们是每个重复序列基质部分中出现的正三联体的一部分；两个基质赖氨酸K179M和K182I，以及螺旋终止负三联体残基E45G、D149S、D249S。在K48A、R152A、D149S和D249S中，细胞ATP合成（OxPhos）几乎完全被抑制，但在K38A中仍为10%，在糖+突变体K179M、K179I + K182I和E45G中为30%至90%。通过ELISA测量的AAC含量与[3H]CAT和[3H]BKA的结合比较显示，K48A、D149S和D249S线粒体存在差异，这证明这些突变在很大程度上消除了抑制剂结合。这些线粒体的OxPhos也无法检测到。与K38A不同，CAT和BKA结合在高AAC水平下得以保留，但OxPhos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。用重组的AAC测量转运活性。在K48A、D149S和D249S中，糖-突变体的电中性ADP/ADP交换在很大程度上或完全被抑制。K38A和R152A仍具有活性，分别为野生型的18%和30%。其他三种交换模式，即ATP/ADP、ADP/ATP和ATP/ATP，在所有糖-突变体中几乎被抑制，但在糖+突变体中仍很高。在糖+中，ATP相关模式高于ADP/ADP模式，但在糖-突变体中较低，导致交换模式反转（EMI）。在对AAC2转运能力的竞争中，较弱的ATP输出模式被强得多的非生产性ADP/ADP模式抑制，从而导致OxPhos受到抑制。与先前的结果一起，现在对三个电荷三联体的所有成员进行了诱变研究，揭示了三个重复结构域内剧烈的功能旋转不对称性。在螺旋内精氨酸三联体中，第三个（R294A）、在正基质三联体中第二个（R152A）以及在螺旋终止负三联体中第一个（E45G）仍表现出高活性。
