Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut, USA.
Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut, USA.
J Biol Chem. 2020 Aug 21;295(34):12305-12316. doi: 10.1074/jbc.RA120.013672. Epub 2020 Jul 10.
Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
Retromer 通过逆行和质膜再循环途径协调从内体中选择和输出完整膜蛋白。关于 retromer 分拣机制的长期假设假设，retromer 与内体膜上相关辅助因子之间的寡聚相互作用驱动 retromer 结合的完整膜货物在其包装到新生运输载体之前聚集。为了检验这个想法，我们使用定量单颗粒荧光显微镜在重组系统中检查了分选连接蛋白 3 (SNX3)-retromer 分选途径的成分之间的相互作用。该系统包括支持的脂质双层、荧光标记的 retromer、SNX3 和两种模型货物蛋白，RAB7 和 WASH 复合物的 WASHC2C 亚基的 retromer 结合片段。我们发现，膜相关 retromer 的分布主要由单体（∼18%）、二聚体（∼35%）、三聚体（∼24%）和四聚体（∼13%）组成。出乎意料的是，膜相关货物的存在或辅助因子都没有显著影响这种分布。结果表明，retromer 在支持的脂质双层上具有形成低阶寡聚体的固有倾向，并且膜结合或辅助因子都不会增强寡聚化。结果支持了这样一种模型，即 SNX3-retromer 是一种适应内体系统中大量膜运输的最低浓缩包被蛋白复合物。
