Prado A, Canal I, Barbas J A, Molloy J, Ferrús A
Instituto Cajal, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
Mol Biol Cell. 1995 Nov;6(11):1433-41. doi: 10.1091/mbc.6.11.1433.
To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.
为了鉴定在体内与肌肉成分相互作用的蛋白质，我们采用了一种基于分离已知肌肉成分突变等位基因抑制子的遗传方法。我们已将此系统应用于果蝇肌钙蛋白I（TnI）及其突变等位基因heldup2（hdp2）的情况。该突变在一个组成型外显子发生单核苷酸变化后，导致第116位的丙氨酸被缬氨酸取代。在分离出的抑制子中，其中一个是由TnI基因本身的第二位点突变引起的。在两个位点都发生突变的TnI的肌肉支持近乎正常的肌原纤维结构，在翅振和飞行测试中表现显著良好，并且分离出的肌纤维产生主动力。我们表明，这种抑制子中的结构和功能恢复并非源于TnI同工型化学计量比的变化。第二位点抑制是由于TnI肌动蛋白结合结构域相邻的七聚体亮氨酸串基序内的亮氨酸被苯丙氨酸取代。这些数据证明了七聚体亮氨酸串在间接飞行肌中具有最显著（即便不是特异性）的结构和功能作用。
