Kölling R, Hollenberg C P
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
EMBO J. 1994 Jul 15;13(14):3261-71. doi: 10.1002/j.1460-2075.1994.tb06627.x.
We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.
我们正在研究多跨膜蛋白Ste6的转运和周转。Ste6蛋白是ABC转运蛋白家族的成员，是酵母交配信息素a因子分泌所必需的。与普遍认为Ste6是一种质膜蛋白的观点相反，我们发现Ste6主要与内膜相关，而不与细胞表面相关。分级分离和免疫荧光数据与Ste6在高尔基体中的定位一致。尽管Ste6主要定位于细胞内，但Ste6蛋白与细胞表面接触，这一发现表明，在胞吞作用突变体中，Ste6在质膜中积累。在胞吞作用突变体中积累在质膜中的Ste6蛋白被泛素化。因此，Ste6是酵母中除MATα2之外第二个已被证明发生泛素化的蛋白。Ste6是一种非常不稳定的蛋白（半衰期为13分钟），在ubc4 ubc5突变体中其稳定性增加约3倍，这表明泛素系统参与了Ste6的降解。然而，在液泡pep4突变体中观察到对Ste6最强的稳定作用（半衰期>2小时），这表明大部分Ste6在液泡中被降解。高效降解Ste6需要分泌功能，这表明Ste6进入分泌途径并通过囊泡载体转运到液泡中。
