Miyazaki Naoyuki, Esaki Masatoshi, Ogura Teru, Murata Kazuyoshi
National Institute for Physiological Sciences, 38 Nishigonaka Myodaiji, Okazaki, Aichi 444-8585, Japan.
Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.
J Struct Biol. 2014 Aug;187(2):187-193. doi: 10.1016/j.jsb.2014.05.010. Epub 2014 Jun 2.
Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.
Cdc48p是一种高度保守的胞质AAA分子伴侣，参与多种细胞过程。它由两个ATP酶结构域（D1和D2）组成，在N端和C端有调节区域。我们最近发现Cdc48p调节线粒体形态，因为D2结构域中ATP酶活性的丧失或正协同作用会导致细胞质中线粒体的严重碎片化和聚集。我们现在使用连续块面扫描电子显微镜（SBF-SEM），一种先进的三维（3D）电子显微镜技术，来检查酿酒酵母中线粒体的结构和形态变化。我们发现缺乏Cdc48p ATP酶活性的突变体表现出线粒体碎片化和聚集，外膜没有融合。这表明Cdc48p的ATP酶活性对于线粒体外膜的融合是必需的。我们的结果还表明，SBF-SEM在对整个细胞中的细胞器和细胞内结构进行形态学和定量研究方面具有相当大的优势。
