Georgiou Xenia, Dimou Sofia, Diallinas George, Samiotaki Martina
Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, Athens 15784, Greece.
Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, Athens 15784, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion 70013, Greece.
Fungal Genet Biol. 2023 Dec;169:103840. doi: 10.1016/j.fgb.2023.103840. Epub 2023 Sep 18.
Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings in Aspergillus nidulans showed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfA GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays.
新合成的质膜（PM）蛋白在共翻译过程中转运至内质网（ER），在内质网出口位点（ERes）富集，并包装进COPII分泌囊泡，通过膜融合被分选至早期高尔基体。高尔基体成熟后，膜货物到达晚期高尔基体，从那里它们以网格蛋白包被的囊泡形式离开，直接或通过内体运往质膜。高尔基体后膜货物运输还涉及细胞骨架和外泌体。高尔基体依赖性分泌途径被认为负责所有主要膜蛋白的运输。然而，我们最近在构巢曲霉中的发现表明，几种质膜货物，如转运蛋白和受体，遵循一种似乎绕过高尔基体功能的分选途径。为了深入了解膜运输，特别是高尔基体旁路，我们在这里使用了邻近依赖性生物素化（PDB）结合数据非依赖型采集质谱（DIA-MS）来鉴定UapA转运蛋白的瞬时相互作用分子。我们的实验包括野生型和影响内质网出口或内吞作用的突变株的蛋白质组分析，确定了可能与UapA运输在生理上相关的预期和新的相互作用。其中，我们使用反向遗传学和荧光显微镜验证了COP I衣被蛋白对于UapA和其他膜货物的内质网出口和正向运输至关重要。我们还表明，ArfA GTP酶激活蛋白（GAP）Glo3在温度升高时有助于UapA的运输。这是第一份使用PDB和蛋白质组学结合真菌遗传学来鉴定膜货物生物发生过程中瞬时相互作用的报告。我们的工作为通过PDB分析剖析动态膜货物运输提供了基础。
