Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic alpha- helix followed by a beta-turn and another alpha-helix, forming a helix- turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resulted in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first alpha-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.
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机译:氨肽酶I(API)是驻留在酵母液泡中的可溶性亮氨酸氨基肽酶(Frey,J。和K.H.Rohm.1978.Biochim.Biophys.Acta.527:31-41)。 API的前体形式包含一个氨基末端的45个氨基酸的前肽,在进入液泡后会被蛋白酶B(PrB)去除。 API的前肽缺乏共有的信号序列,并且已经证明API的液泡定位独立于分泌途径(Klionsky,DJ,R.Cueva和DS Yaver。1992. J. Cell Biol。119:287-299 )。 API前肽的预测二级结构由两亲性α-螺旋,然后是β-螺旋和另一个α-螺旋组成,形成螺旋-螺旋-螺旋结构。通过使用突变分析,我们确定API前肽对于正确转运入液泡至关重要。从API分子中删除整个前肽会导致成熟大小的蛋白质在细胞质中积聚。使用随机诱变和整个原肽中的一系列较小缺失进行的更详细检查显示,API定位受预测的第一个α-螺旋内的变化严重影响。体外研究表明,这种预测的螺旋结构中的突变阻止了生产性结合相互作用的发生。相反,液泡的导入对前肽的第二个预测螺旋的变化相对不敏感。对API折叠的检查表明,影响进入液泡的突变不影响API的结构。这些数据表明API前肽在沿细胞质至液泡靶向途径的关键步骤用作液泡靶向决定簇。
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