The Infrastructure of Mitochondrial Matrix Proteins

线粒体基质蛋白的基础设施

• 批准号: 9418565
• 负责人: Paul Srere
• 金额: $ 30万
• 依托单位: University of Texas Southwestern Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9418565&HistoricalAwards=false
• 关键词: Infrastructure; Mitochondrial; Matrix; Proteins

Srere 9418565 In order to study the problem of metabolite channelling, a recent and elegant technique employed has been to link the DNA for two enzymes and to express the fused protein in cells. We have used this latter technique to prepare a fusion protein of yeast mitochondrial citrate synthase (CS1) yeast mitochondrial malate dehydrogenase (MDH1). Yeast cells which lack mitochondrial enzyme activity of the Krebs TCA cycle cannot grow on acetate (acetate-phenotype). When either CS1 or MDH1 is deleted from yeast cells (CS1- or MDH1- cells) or when a double deletion is made (CS1-/MDH1- cells) the cells cannot grow on acetate. The fusion protein restored growth on acetate when placed in yeast cells which lacked CS1, MDH1, and CS1 and MDH1. In addition, kinetic studies on the purified fusion protein indicated channelling of the intermediate oxalacetate (OAA). We propose to extend these studies by changing the order of the enzymes; by testing linkers of different lengths and sequences; and by using heterologous enzymes of CS and MDH. Nonsequential fusion proteins of Krebs TCA cycle enzymes will be made also. We will investigate the in vitro channeling assay using different OAA trapping systems and isotopic methods. %%% When cells are broken, one finds apparently mainly free enzymes which given rise to the idea that the cell is essentially a "bag of enzymes". There are many indications, however, that in the cell, sequential metabolic enzymes are in complexes which dissociate upon cell rupture. We have studied the interactions of sequential enzymes and their consequences in a variety of ways, including biophysical and genetic techniques. This grant proposal is to continue our studies on a fused two sequential enzyme systems, yeast mitochondrial malate dehydrogenase (MDH), and yeast mitochondrial citrate synthase (CS). This fusion protein was isolated and its kinetic studied. The results indicated that channeling of the intermediate occurred. We propose to study the phenomenon by producing a series of other fusion proteins of MDH and CS with changed order, changed linkers, and heterologous MDH and CS. These studies will serve as models for possible metabolic advantages and regulatory properties of enzyme-enzyme interactions in cells. ***

为了研究代谢物通道的问题，最近采用的一种先进技术是连接两种酶的DNA并在细胞中表达融合蛋白。 我们已经使用后一种技术来制备酵母线粒体柠檬酸合酶（CS 1）和酵母线粒体苹果酸脱氢酶（MDH 1）的融合蛋白。 缺乏Krebs TCA循环的线粒体酶活性的酵母细胞不能在乙酸盐上生长（乙酸盐表型）。 当CS 1或MDH 1从酵母细胞中缺失（CS 1-或MDH 1-细胞）或当进行双缺失（CS 1-/MDH 1-细胞）时，细胞不能在乙酸盐上生长。 当将融合蛋白置于缺乏CS 1、MDH 1以及CS 1和MDH 1的酵母细胞中时，该融合蛋白在乙酸盐上恢复生长。 此外，对纯化的融合蛋白的动力学研究表明，中间产物乙酸乙酯（OAA）的通道。 我们建议通过改变酶的顺序，通过测试不同长度和序列的接头，以及通过使用CS和MDH的异源酶来扩展这些研究。 还将制备Krebs TCA循环酶的非顺序融合蛋白。 我们将使用不同的OAA捕获系统和同位素方法研究体外通道分析。 当细胞破碎时，人们发现显然主要是游离的酶，这引起了细胞本质上是一个“酶袋”的想法。 然而，有许多迹象表明，在细胞中，顺序代谢酶是在细胞破裂时解离的复合物中。 我们已经研究了顺序酶的相互作用及其后果，在各种方式，包括生物物理和遗传技术。 本研究计画将继续我们对酵母线粒体苹果酸脱氢酶（MDH）与酵母线粒体柠檬酸合酶（CS）两个连续酵素系统的研究。 分离该融合蛋白并研究其动力学。 结果表明，中间体发生沟流现象。 我们建议研究 通过产生一系列顺序改变、接头改变的MDH和CS的其他融合蛋白以及异源MDH和CS来实现该现象。 这些研究将作为可能的代谢优势和细胞中酶-酶相互作用的调节特性的模型。 ***