Singer-Krüger B, Frank R, Crausaz F, Riezman H
Biocenter, University of Basel, Switzerland.
J Biol Chem. 1993 Jul 5;268(19):14376-86.
Previously (Singer, B., and Riezman, H. (1990) J. Cell Biol. 110, 1911-1922), we provided evidence for the existence of an endocytic intermediate(s) from the yeast Saccharomyces cerevisiae that is responsible for the transport of the pheromone alpha-factor from the plasma membrane to the vacuole. Here we show by kinetic analysis that the endocytic apparatus of yeast is composed of early and late endosomes, similar to what has been found in animal cells. We have developed a three-step isolation procedure to purify early and late endosomes, consisting of differential centrifugation, flotation on a Nycodenz density gradient, and sedimentation density gradient centrifugation on sucrose/D2O. Using internalized 35S-alpha-factor as a marker, the endosomal fractions were substantially enriched over other membranes, except for Golgi elements and a compartment containing binding protein. These contaminants could not be removed by other standard purification methods. We have analyzed the protein composition of our most pure early and late endosome fractions. By two-dimensional gel analysis we identified more than 20 proteins spots that are highly enriched in the early/late endosomal fractions. N-terminal protein sequencing resulted in the identification of four novel proteins.
此前（Singer, B.和Riezman, H.（1990年）《细胞生物学杂志》110卷，1911 - 1922页），我们提供了证据，证明酿酒酵母存在一种内吞中间体，其负责将信息素α-因子从质膜运输至液泡。在此我们通过动力学分析表明，酵母的内吞装置由早期和晚期内体组成，这与在动物细胞中发现的情况类似。我们开发了一种三步分离程序来纯化早期和晚期内体，包括差速离心、在Nycodenz密度梯度上漂浮以及在蔗糖/D₂O上进行沉降密度梯度离心。以内化的³⁵S-α-因子作为标记物，除了高尔基体成分和一个含有结合蛋白的区室之外，内体组分相对于其他膜有显著富集。这些污染物无法通过其他标准纯化方法去除。我们分析了最纯的早期和晚期内体组分的蛋白质组成。通过二维凝胶分析，我们鉴定出20多个在早期/晚期内体组分中高度富集的蛋白质斑点。N端蛋白质测序鉴定出了四种新蛋白质。
