ISOZYMES OF ASPARTATE TRANSAMINASE

在这个建议中，我们专注于探索两种蛋白质的折叠， 天冬氨酸氨基转移酶的同工酶，由核蛋白编码， 在细胞质游离多聚核糖体中合成。 其中一 蛋白质（胞质，cAAT）保留在细胞质区室中， 另一个（线粒体，mAAT）最终居住在 线粒体基质 此外，合成mAAT作为前体 蛋白（pmAAT）与额外的氨基末端延伸称为 前序列或信号肽。 分子机制， 蛋白质的细胞内靶向和易位已经被积极地 调查，但很少有人知道的早期阶段， 可能是由编码在氨基酸中的信息决定的 酸性序列 越来越多的证据表明分子伴侣， 一组在所有类型的细胞中普遍存在的蛋白质， 控制这些事件。 我们的主要目标是阐明 蛋白质在给定细胞内区室中的折叠取决于 氨基酸序列中携带的信息， 分子伴侣存在于该隔室中或两者上。 的 直接目标是：1）描述a）前序列的作用 pmAAT中的肽和B）成熟cAAT和mAAT中的其他确定片段 在这些蛋白质的折叠过程中，当它单独在缓冲液中发生时（在 体外）和在细胞质提取物存在下（原位条件）。 这项分析将利用嵌合的工程蛋白质， 信号肽与胞质同工酶的融合或通过交换 两种同工酶之间的一些相关区域的片段。 2)分析 野生型和嵌合形式的相互作用在目的1中提到 与特定的伴侣（从Grovel和hsp70开始），以确定 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.