Analysis of common molecular basis of the AAA ATPase

AAA家族蛋白的AAA ATP酶结构域的分子机制如ATP水解和底物蛋白的处理已经被研究。AAA蛋白酶FtsH的六元ATP酶环的孔区域周围的残基的诱变，表明一些酸性残基在功能上是重要的。我们发现FtsH及其C. elegans同源物不需要ATP水解，并且它也不依赖于位于孔处的保守芳香残基，这表明能量不依赖性底物易位的可能性。我们利用C. elegans fidgetin同系物，FIGL-1。我们观察到CDC-48.1和CDC-48.2、p97在C. elegans以ATP非依赖性方式抑制亨廷顿蛋白片段的聚集体形成。SPAS-1、C. elegans的spastin同源物和人katanin对微管动力学的影响。野生型SPAS-1的过表达引起微管网络的解体，而步行者或孔突变体则没有。另一方面，在荧光显微镜下观察人katanin对荧光标记的微管的切断。结果发现，katanin的孔突变体失去了微管切割活性。在高速原子力显微镜下观察到依赖于katanin和ATP的微管断裂过程。

Molecular mechanisms of the AAA ATPase domain of AAA family proteins such as ATP hydrolysis and handling of substrate proteins have been studied. Mutagenesis of residues around the pore region of the hexametric ATPase ring of an AAA protease, FtsH, indicated that some acidic residues are functionally important. We have found that degradation of unfolded polypeptides by some chimeras of FtsH and its C. elegans homologs did not require ATP hydrolysis, and that it was also independent of the conserved aromatic residues located at the pore, suggesting the possibility of energy-independent substrate translocation. We have obtained firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases using mutants of the C. elegans fidgetin homolog, FIGL-1. We have observed that CDC-48.1 and CDC-48.2, p97 homologs in C. elegans, suppress aggregate formation of huntingtin fragments in an ATP-independent manner. Effects of SPAS-1, the C. elegans homolog of spastin, and human katanin on microtubule dynamics have been studied. Overexpression of wild-type SPAS-1 caused disassembly of microtubule network, whereas that of a Walker or pore mutant did not. On the other hand, severing of fluorescently labeled microtubules by human katanin was observed under a fluorescent microscope. It was found that pore mutants of katanin lost the microtubule-severing activity. Processes of microtubule-severing, which was dependent on both katanin and ATP, were observed under a high-speed atomic force microscope.