FISSION PROTEIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 迄今为止，在酵母的分裂过程中已经鉴定出三种蛋白质：Fis 1、Dnm 1和Mdv 1。Fis 1通过C-末端跨膜结构域（尾部锚定蛋白）靶向细胞器膜。Dnm 1是一种被认为是机械酶的大的GTd 3，属于发动蛋白超家族。Mdv 1是一种衔接蛋白。目前我们正在测试的模型是，Fis 1招募GTd 3 Dnm 1的网站膜断裂和Mdv 1的行为，以进一步刺激Dnm 1组装，GTP水解，和膜断裂。我们想补充我们的生物化学和NMR/X射线结构的研究与单粒子重建和层析成像研究这个系统。一个目标是建立裂变机制的结构模型，作为理解这些蛋白质如何将GTP水解与膜断裂偶联的一步。 我们已经获得了与N-末端结构域（1-236）和具有卷曲螺旋结构域的N-末端结构域（1-302）的麦芽糖结合蛋白融合的Mdv 1构建体的良好表达和纯化。 MBP-Mdv 1（1-302）似乎是自组装的，负染色EM显示可能来自MBP或Mdv 1本身的原丝。该提案专门用于对我们的MBPMdv 1（1-302）样品进行成像，然而，我们认为在该项目上需要与NCMI长期合作，因为我们在膜和其他组件的存在下构建系统。 我们试图确定线粒体和过氧化物酶体分裂的分子基础，这是细胞器功能所必需的过程，对人类健康至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To date, three proteins have been identified in the fission process in yeast: Fis1, Dnm1, and Mdv1. Fis1 is targeted to the organellar membrane by a C-terminal transmembrane domain (a tail-anchored protein). Dnm1 is a large GTPase thought to act as a mechanoenzyme and belongs to the dynamin superfamily. Mdv1 is an adaptor protein. The current model we are testing is that Fis1 recruits the GTPase Dnm1 to sites of membrane scission and Mdv1 acts to further stimulate Dnm1 assembly, GTP hydrolysis, and membrane scission. We would like to complement our biochemical and NMR/x-ray structural studies with single particle reconstruction and tomography studies on this system. One goal is to build structural models of the fission machinery as a step towards understanding how these proteins couple GTP hydrolysis to membrane scission. We have obtained good expression and purification of Mdv1 constructs fused to maltose binding protein of the N-terminal domain (1-236) and the N-terminal domain with the coiled coil domain (1-302). MBP-Mdv1(1-302) appears to self-assemble, and negative stain EM reveals protofilaments that may arise from MBP, or from Mdv1 itself. This proposal is specifically to image our MBPMdv1(1-302) sample, however, we see a long-term need to work with NCMI on this project as we build up the system in the presence of membranes and other components. We seek to determine the molecular basis for mitochondrial and peroxisome fission, a process essential for organellar function and important in human health.