Liu Hao-Yang, Ashby Grant, Yuan Feng, Sarkar Susovan, Hayden Carl C, Huibregtse Jon M, Stachowiak Jeanne C
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, United States.
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, United States.
bioRxiv. 2024 Dec 18:2024.12.17.628947. doi: 10.1101/2024.12.17.628947.
Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation.
Ubiquitylation is essential for maintaining cellular homeostasis by regulating transmembrane protein trafficking, degradation, and signaling. Traditionally, poly-ubiquitylation has been viewed primarily as a signal for protein degradation, while mono-ubiquitylation is considered sufficient to trigger endocytosis. However, our work reveals a previously unrecognized role for poly-ubiquitylation in clathrin-mediated endocytosis, demonstrating that poly-ubiquitylated transmembrane proteins outcompete their non-ubiquitylated counterparts for incorporation into clathrin-coated vesicles, thereby establishing a competitive framework for endocytic cargo sorting. This mechanism reveals a selective sorting mechanism driven by the extent of ubiquitylation, which could regulate the removal of damaged proteins while protecting functional proteins at the plasma membrane.
跨膜蛋白的内吞循环对于细胞信号传导、配体摄取、蛋白质运输和降解至关重要。许多跨膜蛋白的胞内结构域会发生泛素化，这通过网格蛋白介导的内吞作用促进其内化。这种增强的内化作用会如何影响缺乏泛素化的跨膜蛋白的内吞摄取呢？最近的研究表明，多种跨膜蛋白在高度拥挤的内吞结构中竞争空间，这表明一组跨膜蛋白的增强内化可能是以其他组为代价的。在这里，我们表明多泛素化跨膜蛋白的优先内化导致单泛素化和非泛素化蛋白的内吞作用减少。通过结合活细胞成像和配体摄取分析，我们证实泛素化增加与网格蛋白包被小泡的内化增加相关。此外，多泛素化受体在定位到内吞位点和摄取细胞外配体方面显著胜过其单泛素化和非泛素化的对应物。这些发现证明了跨膜蛋白循环中固有的相互依赖性，表明网格蛋白包被小泡充当选择性过滤器，优先摄取高度泛素化的跨膜蛋白，而将很少或没有泛素化的蛋白留在后面。鉴于多泛素化被认为是蛋白质老化和损伤的信号，我们的发现提出了一种高优先级货物蛋白选择性内化的机制，同时排除和保护缺乏多泛素化的功能蛋白。
泛素化通过调节跨膜蛋白运输、降解和信号传导对于维持细胞内稳态至关重要。传统上，多泛素化主要被视为蛋白质降解的信号，而单泛素化被认为足以触发内吞作用。然而，我们的工作揭示了多泛素化在网格蛋白介导的内吞作用中以前未被认识的作用，表明多泛素化跨膜蛋白在并入网格蛋白包被小泡方面胜过其非泛素化对应物，从而建立了内吞货物分选的竞争框架。这种机制揭示了一种由泛素化程度驱动的选择性分选机制，它可以调节受损蛋白的去除，同时保护质膜上的功能蛋白。
