Oda M N, Scott S V, Hefner-Gravink A, Caffarelli A D, Klionsky D J
Section of Microbiology, University of California, Davis 95616, USA.
J Cell Biol. 1996 Mar;132(6):999-1010. doi: 10.1083/jcb.132.6.999.
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.
氨肽酶I（API）是一种存在于酵母液泡中的可溶性亮氨酸氨肽酶（弗雷，J.，和K.H.罗姆。1978年。《生物化学与生物物理学报》。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，在进入液泡时被蛋白酶B（PrB）去除。API的前肽缺乏一致的信号序列，并且已经证明API的液泡定位独立于分泌途径（克利昂斯基，D.J.，R.库埃瓦，和D.S.亚弗。1992年。《细胞生物学杂志》。119:287 - 299）。API前肽的预测二级结构由一个两亲性α - 螺旋、一个β - 转角和另一个α - 螺旋组成，形成一个螺旋 - 转角 - 螺旋结构。通过突变分析，我们确定API前肽对于正确转运到液泡中是必不可少的。从API分子中删除整个前肽导致成熟大小的蛋白质在细胞质中积累。使用随机诱变和前肽中一系列较小的缺失进行的更详细检查表明，API的定位受到预测的第一个α - 螺旋内改变的严重影响。体外研究表明，这个预测螺旋中的突变会阻止有效的结合相互作用发生。相比之下，液泡导入对前肽预测的第二个螺旋中的改变相对不敏感。对API折叠的检查表明，影响进入液泡的突变不会影响API的结构。这些数据表明，API前肽在从细胞质到液泡的靶向途径的关键步骤中作为液泡靶向决定因素。
