ISOZYMES OF ASPARTATE TRANSAMINASE

在这个提案中，我们专注于探索两种蛋白质的折叠， 天冬氨酸氨基转移酶同工酶，由核编码 基因组，并在细胞质游离多聚体中合成。这其中的一个 蛋白质(胞浆，CAAT)保留在细胞质隔间，用于其 整个生命周期；另一个(线粒体，MAT)最终居住在 线粒体基质。此外，还合成了MAAT作为前驱体 蛋白质(PmAAT)，带有一个额外的氨基末端延伸，称为 前序或信号肽。人类免疫缺陷的分子机制 蛋白质在细胞内的靶向和转位一直很活跃 进行了调查，但对早期阶段的了解很少 排序可能由编码在氨基中的信息决定 酸序列。越来越多的证据表明，分子伴侣， 在所有类型的细胞中普遍存在的一组蛋白质，可能有一定的作用 对这些事件的控制。我们的主要目标是澄清 蛋白质在特定细胞内的折叠取决于 在特定集合上，在其氨基酸序列中携带的信息 分子伴侣存在于该隔间或两个隔间。这个 直接目标是：1)描述a)前置序列的作用 成熟CAAT和MAAT中pmAAT中的多肽和其他已定义片段的b) 在这些蛋白质的折叠过程中，因为它只出现在缓冲液中(在 体外)和存在细胞质提取液(原位条件)。 这项分析将利用嵌合的、由 信号肽与胞浆同工酶的融合或通过交换 两个同工酶之间的几个相关区域的片段。2)分析 目标1中提到的野生型和嵌合体的相互作用 使用特定的伴侣(以Grovel和HSP70开始)，以识别 蛋白质在复合体中的接触位置和折叠状态。

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.