De Marcos Lousa Carine, Trézéguet Véronique, Dianoux Anne-Christine, Brandolin Gérard, Lauquin Guy J-M
Laboratoire de Physiologie Moléculaire et Cellulaire, Institut de Biochimie et Génétique Cellulaires, 1, rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France.
Biochemistry. 2002 Dec 3;41(48):14412-20. doi: 10.1021/bi0261490.
The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.
线粒体腺嘌呤核苷酸载体，即Ancp，在维持真核细胞的能量通量方面发挥着关键作用。已发现人类疾病与异常的人类ANC基因（HANC）表达有关，也与该蛋白的直接失活有关，失活原因要么是自身抗体结合，要么是突变。然而，三种HAncp同工型的个体生化特性尚未得到解析。为此，在ScANC2调控序列的控制下，三种HANC开放阅读框在酵母中得以表达。三种HANC中的每一种都能够恢复缺乏其三种内源性ANC的酵母突变株在非发酵碳源上的生长。然后，它们的ADP/ATP交换特性首次在分离的线粒体中得以测量。HANC3在恢复酵母生长方面效率最高，且HAnc3p呈现出最高的V（M）（80 nmol ADP min⁻¹ mg蛋白⁻¹）和K（ADP）（M）（8.4 μM）。HAnc1p和HAnc2p呈现出相似的动力学常数（V（M）约为30 - 40 nmol ADP min⁻¹ mg蛋白⁻¹，K（ADP）（M）约为2.5 - 3.7 μM），其数值与HANC1和HANC2恢复酵母生长的较低能力相一致。然而，HANC基因恢复生长的水平低于ScANC2，这表明HAncp的量在体内可能是有限的。为了优化HAncp的产生，我们通过对HAnc1p N端的两个带电荷氨基酸进行诱变来研究它们在线粒体中的生物合成。观察到D3A和D3K突变产生了严重影响，阻止了酵母生长。相反，与野生型相比，K10A突变增加了酵母生长互补性和核苷酸交换率。这些结果表明HAnc1p的N端区域对其在酵母线粒体中的生物合成和转运活性具有重要意义。
