ISOZYMES OF ASPARTATE TRANSAMINASE

在这个提案中，我们重点探索两种蛋白质的折叠， 天冬氨酸转氨酶的同工酶，由核编码 基因组并在无细胞质的多聚核糖体中合成。 其中之一 蛋白质（胞质，cAAT）保留在细胞质区室中 整个生命周期；另一个（线粒体，mAAT）最终驻留在 线粒体基质。 此外，mAAT 作为前体被合成 蛋白质 (pmAAT) 具有一个额外的氨基末端延伸，称为 前序列或信号肽。 的分子机制 蛋白质的细胞内靶向和易位已被积极研究 进行了调查，但对早期阶段知之甚少 排序可能是由氨基编码的信息决定的 酸序列。 越来越多的证据表明分子伴侣， 一组普遍存在于所有类型细胞中的蛋白质，可能发挥作用 在这些事件的控制中。 我们的主要目标是阐明是否 蛋白质在给定细胞内区室中的折叠取决于 特定集合上其氨基酸序列中携带的信息 存在于该隔室或两者上的分子伴侣。 的 近期目标是： 1) 描述 a) 前序的作用 pmAAT 中的肽和 b) 成熟 cAAT 和 mAAT 中其他定义片段 在这些蛋白质的折叠过程中，当它发生在单独的缓冲液中时（在 体外）和细胞质提取物存在下（原位条件）。 该分析将利用由以下方法制备的嵌合工程蛋白质 信号肽与胞质同工酶融合或通过交换 两个同工酶之间的一些相关区域的片段。 2）分析 目标 1 中提到的野生型和嵌合型的相互作用 与特定的伴侣（从 Grovel 和 hsp70 开始），以识别 复合物中蛋白质的接触位点和折叠状态。

In this proposal we focus on exploring the folding of two proteins, the isozymes of aspartate aminotransferase, which are encoded by the nuclear genome and synthesized in cytoplasmic free polysomes. One of these proteins (cytosolic, cAAT) remains in the cytoplasmic compartment for its whole life span; the other (mitochondrial, mAAT) ends up residing in the mitochondrial matrix. Furthermore, mAAT is synthesized as a precursor protein (pmAAT) with an additional amino terminal extension known as the presequence or signal peptide. The molecular mechanisms for the intracellular targeting and translocation of proteins have been actively investigated, but very little is known regarding the early stages of sorting which are probably dictated by information encoded in the amino acid sequence. Mounting evidence indicates that molecular chaperones, a group of proteins ubiquitous in all types of cells, might have a role in the control of these events. Our main goal is to elucidate whether the folding of a protein in a given intracellular compartment depends on the information carried in its amino acid sequence, on the particular set of molecular chaperones present in that compartment or on both. The immediate aims are: 1) Characterize the role of a) the presequence peptide in pmAAT and b) of other defined segments in mature cAAT and mAAT in the folding process of these proteins as it occurs in buffer alone (in vitro) and in the presence of cytoplasmic extracts (in situ conditions). This analysis will make use of chimeric, engineered proteins prepared by fusion of the signal peptide to the cytosolic isozyme or by exchanging segments of a few pertinent regions between the two isozymes. 2) Analyze the interaction of the wild type and chimeric forms alluded to in aim 1 with specific chaperones (starting with Grovel and hsp70), to identify contact sites and folding states of the proteins in the complexes.