Nishikawa S, Umemoto N, Ohsumi Y, Nakano A, Anraku Y
Department of Biology, Faculty of Science, University of Tokyo, Japan.
J Biol Chem. 1990 May 5;265(13):7440-8.
To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.
为了研究酵母液泡的生物发生过程，我们寻找了定位于液泡膜的新型标记蛋白。通过伴刀豆球蛋白A-琼脂糖柱层析从液泡膜囊泡中制备糖蛋白，并用于制备单克隆抗体。获得的抗体识别几种具有N-连接寡糖链的液泡蛋白。一组抗体与一种主要分子质量为72 kDa的液泡糖蛋白（vgp72）发生反应，该蛋白似乎在外周与液泡膜结合。通过脉冲追踪实验以及使用各种分泌突变体（sec18、sec7和sec1）和液泡蛋白酶突变体（pep4）进行分析，对vgp72的生物合成进行了详细研究。vgp72最初以内质网中74 kDa的形式出现，并在高尔基体中迅速被修饰为两种不同的形式：一种79 kDa的形式和一种糖基化程度不均一的形式（90 - 150 kDa）。随后，这两种形式在液泡中都经过蛋白水解加工，产生了72 kDa的形式以及高度糖基化的形式。因此，vgp72的生物发生过程利用了分泌途径的早期部分，就像液泡可溶性酶的情况一样。一个独特的特征是，两种糖基化程度不同的形式似乎遵循相同的路径到达液泡膜。
