TOMOGRAPHY OF YEAST MITOCHONDRIA THAT HAVE UNDERGONE PERMEABILITY TRANSITION

(由NIH HL49182至D.R.Pfeiffer支持)酵母线粒体 (酿酒酵母)含有渗透性转换孔 它的调节方式与哺乳动物的毛孔不同 线粒体。在含有10 mMPI和乙醇的甘露醇介质中 (可氧化底物)，酵母线粒体积累了大量的 当加入一个电泳性钙离子载体时，钙离子发生变化。毛孔 即使在Ru的作用下，钙摄取后也不会发生开放 红抑制条件下大鼠肝线粒体快速开孔 相似的条件。然而，酵母中确实会出现毛孔。 线粒体，当不存在钙和磷时，由 肿胀、超微结构和基质溶质释放。孔洞张开是 缓慢，除非提供呼吸底物(乙醇或NADH)，但 当有寡霉素存在时，也会迅速对三磷酸腺苷做出反应。 等渗聚乙二醇致酵母收缩 线粒体在呼吸过程中或在AT-P存在时肿胀， 当溶质大小为1.0-1.1 kDa时，50%有效。在这一特殊情况下 研究，我们希望看到酵母的超微结构变化 线粒体处于收缩状态、诱导肿胀状态(通过 添加乙醇)，然后处于由 添加聚乙二醇酯。厚层切片的层析重建是 被用来帮助我们分析结构的变化 线粒体在所有三个阶段都有隆起。七个倾斜系列是 使用HVEM记录在0.5(m厚的塑料切片-嵌入 分离的酵母线粒体。三个重建和模型是 由对照(收缩的)线粒体和一个重新收缩的线粒体组成 线粒体。高中生艾丽斯·叶正在协助这项工作 西屋科学竞赛的项目。薄层EM切片 对照、肿胀和重新收缩的酵母线粒体 拍摄以记录每个种群内的变异性。这个 重建的模型显示，在收缩和 再收缩的线粒体，在晶状体内间隙和 内膜和外膜之间的空间被压缩， 尽管狭窄不具有相同的长、管状 与其他线粒体(大鼠肝脏、神经元、肌肉、 和粗糙脉孢菌)。初步结果是 在奥尔巴尼线粒体研究前沿会议上的展示 在九月份。Mannella，C.A.，Buttle，K.，Pfeiffer，D.R.，Rath，B.K.， Marko，M.(1998)线粒体设计：对 渗透率转变的结构基础。程序奥尔巴尼 线粒体研究前沿会议。

(Supported by NIH HL49182 to D..R. Pfeiffer) Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ upon the addition of an electrophoretic Ca2+ ionophore. Pore opening does not occur following Ca2+ uptake even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH), but also occurs rapidly in response to ATP when oligomycin is present. Iso-osmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the pres ence of AT P, is 50% effective at a solute size of 1.0-1.1 kDa. In this particular study, we wish to see the ultrastructural changes of yeast mitochondria in a contracted state, an induced swollen state (by addition of ETOH) and then in a recontracted state induced by the addition of PEG. Tomographic reconstructions from thick sections are being used to help us to analyze the changes in structure of the mitochondrial cristae in all three phases. Seven tilt series were recorded using the HVEM, on 0.5(m-thick sections of plastic-embedded isolated yeast mitochondria. Three reconstructions and models were made of control (contracted) mitochondria and one of a recontracted mitochondrion. A high school student, Alice Yip, is assisting on this project for the Westinghouse Science Competition. Thin EM sections of control, swollen, and recontracted yeast mitochondria were photographed to document the variability within each population. The models from the reconstructions showed that in both contracted and recontracted mitochondria, the flow between the intracristal space and the space between the inner and outer membranes is constricted, although the constrictions do not have the same long, tubular appearance as those in other mitochondria (rat liver, neurons, muscle, and Neurospora crassa) we have studied. Preliminary results were shown at the Albany Conference on Frontiers of Mitochondrial Research in September. Mannella, C.A., Buttle, K., Pfeiffer, D.R., Rath, B.K., Marko, M. (1998) Mitochondrial design: implications for the structural basis of the permeability transition. Proc. Albany Conference on Frontiers of Mitochondrial Research.