Department of Biology/Chemistry, Molecular Membrane Biology Group, University of Osnabrück, Osnabrück, Germany.
Center of Cellular Nanoanalytics, University of Osnabrück, Osnabrück, Germany.
Elife. 2019 Jan 29;8:e42837. doi: 10.7554/eLife.42837.
Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. Previously we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.
质膜的蛋白质和脂质通过分泌途径和内吞途径的结合进行持续重塑。在酵母内吞途径中，货物被分拣用于回收至质膜或在液泡中降解。之前我们已经表明 GARP 复合物在鞘脂代谢平衡和分拣中起作用（Fröhlich 等人，2015）。然而，在 GARP 依赖的过程中分拣的大多数货物仍然知之甚少。在这里，我们使用生长素诱导的 GARP 降解与基于质谱的液泡蛋白质组学和脂质组学相结合，表明两种特定类型蛋白质（氨基磷脂翻转酶和细胞壁合成蛋白）的回收依赖于功能正常的 GARP 复合物。我们的结果表明，翻转酶的分拣错误和脂质组成的重塑是 GARP 突变体中最早发生的缺陷。我们的测定方法可以适应系统地绘制整个内吞途径的货物图谱。
