Losko S, Kopp F, Kranz A, Kölling R
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Mol Biol Cell. 2001 Apr;12(4):1047-59. doi: 10.1091/mbc.12.4.1047.
Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.
先前的实验表明，酿酒酵母的α-因子转运蛋白Ste6向酵母液泡的运输受泛素化调节。为了确定运输途径中依赖泛素化的步骤，我们通过免疫荧光实验、使用Ste6-绿色荧光蛋白融合蛋白以及蔗糖密度梯度分级分离法，研究了泛素化缺陷型doa4突变体中Ste6的细胞内定位。我们发现，Ste6在doa4突变体的液泡膜上积累，而不在细胞表面积累。对doa4 pep4双突变体的实验表明，doa4突变体中Ste6进入液泡腔的过程受到抑制。额外表达泛素可以抑制摄取缺陷，这表明它主要是泛素水平降低（从而泛素化减少）的结果，而不是去泛素化缺陷的结果。基于我们的发现，我们提出，Ste6或运输因子的泛素化是Ste6分选进入多泡体途径所必需的。此外，我们获得的证据表明，Ste6在一个内部区室和质膜之间循环。
