Bhar Debjani, Karren Mary Anne, Babst Markus, Shaw Janet M
Biochemistry Department, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650.
Biology Department, University of Utah, Salt Lake City, Utah 84112-0840.
J Biol Chem. 2006 Jun 23;281(25):17312-17320. doi: 10.1074/jbc.M513530200. Epub 2006 Apr 6.
Interactions between yeast Dnm1p, Mdv1p, and Fis1p are required to form fission complexes that catalyze division of the mitochondrial compartment. During the formation of mitochondrial fission complexes, the Dnm1p GTPase self-assembles into large multimeric complexes on the outer mitochondrial membrane that are visualized as punctate structures by fluorescent labeling. Although it is clear that Fis1p.Mdv1p complexes on mitochondria are required for the initial recruitment of Dnm1p, it is not clear whether Dnm1p puncta assemble before or after this recruitment step. Here we show that the minimum oligomeric form of cytoplasmic Dnm1p is a dimer. The middle domain mutant protein Dnm1G385Dp forms dimers in vivo but fails to assemble into punctate structures. However, this dimeric mutant stably interacts with Mdv1p on the outer mitochondrial membrane, demonstrating that assembly of stable Dnm1p multimers is not required for Dnm1p-Mdv1p association or for mitochondrial recruitment of Dnm1p. Dnm1G385Dp is reported to be a terminal dimer in vitro. We describe conditions that allow assembly of Dnm1G385Dp into functional fission complexes on mitochondria in vivo. Using these conditions, we demonstrate that multimerization of Dnm1p is required to promote reorganization of Mdv1p from a uniform mitochondrial localization into punctate fission complexes. Our studies also reveal that Fis1p is present in these assembled fission complexes. Based on our results, we propose that Dnm1p dimers are initially recruited to the membrane via interaction with Mdv1p.Fis1p complexes. These dimers then assemble into multimers that subsequently promote the reorganization of Mdv1p into punctate fission complexes.
酵母Dnm1p、Mdv1p和Fis1p之间的相互作用对于形成催化线粒体区室分裂的裂变复合体是必需的。在线粒体裂变复合体形成过程中，Dnm1p GTP酶在线粒体外膜上自组装成大型多聚体复合体，通过荧光标记可将其可视化为点状结构。虽然很明显线粒体上的Fis1p.Mdv1p复合体是Dnm1p初始募集所必需的，但尚不清楚Dnm1p点状结构是在该募集步骤之前还是之后组装。在这里，我们表明细胞质Dnm1p的最小寡聚形式是二聚体。中间结构域突变蛋白Dnm1G385Dp在体内形成二聚体，但无法组装成点状结构。然而，这种二聚体突变体与线粒体外膜上的Mdv1p稳定相互作用，表明稳定的Dnm1p多聚体的组装对于Dnm1p-Mdv1p结合或Dnm1p的线粒体募集不是必需的。据报道，Dnm1G385Dp在体外是末端二聚体。我们描述了在体内使Dnm1G385Dp组装成线粒体上功能性裂变复合体的条件。利用这些条件，我们证明Dnm1p的多聚化是促进Mdv1p从均匀的线粒体定位重组成点状裂变复合体所必需的。我们的研究还揭示Fis1p存在于这些组装好的裂变复合体中。基于我们的结果，我们提出Dnm1p二聚体最初通过与Mdv1p.Fis1p复合体相互作用被募集到膜上。然后这些二聚体组装成多聚体，随后促进Mdv1p重组成点状裂变复合体。
