Journal Article

A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes

Gschweitl, Michaela, Huotari, Jatta, Yamauchi, Yohei, Stoffel-Studer, Ingrid, Barnes, Christopher A, Greber, Urs F, Meyer-Schaller, Nathalie, Enchev, Radoslav I, Ulbricht, Anna, Helenius, Ari, Peter, Matthias

2016

Overview

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. Individual cells can move material, collectively referred to as cargo, from the outside environment into the cell interior via a process known as endocytosis. The cell then has different routes to transport the packages of cargo, called endocytic vesicles, to specific locations within the cell. Protein-based molecular machines move the cargo and control how it is selected and targeted to different destinations. For example, a molecular machine that contains a protein called CUL3 labels other components of the system with a chemical tag to regulate the route cargo takes in mammalian cells. However, it was not clear how CUL3 can selectively attach the chemical labels. Gschweitl, Ulbricht et al. have now found that another protein called SPOPL provides selectivity for the CUL3-based machine during endocytosis in human cells. The experiments show that SPOPL attaches to endocytic vesicles, and that CUL3 and SPOPL work together to label a specific component of these vesicles called EPS15. The label changes how EPS15 interacts with other proteins. When SPOPL is not present in a cell, EPS15 is unnaturally stable and occupies many of the routes used by endocytic cargos. The cargo directly interacting with EPS15 is then routed on the fast lane to its destination, while other cargo accumulate in a kind of molecular traffic jam. Other proteins like SPOPL are specific for the endocytic system. Exchange of SPOPL with these similar proteins in the CUL3 machine is likely to chemically label a different set of endocytic proteins. Gschweitl, Ulbricht et al.’s next challenge is to identify the selectivity, targeting and coordination of these exchangeable components in the endocytic system.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Adaptor Proteins, Vesicular Transport - metabolism / Biochemistry / Biological Transport / Calcium-Binding Proteins - metabolism / Cell Biology / Cell Line / CUL3 / Cullin / Cullin Proteins - metabolism / EGFR / Endocytosis / Endosomes / Endosomes - metabolism / Enzymes / Epidermal growth factor receptors / EPS15 / Humans / Infections / Influenza / Influenza A / influenza A virus / Influenza A virus - physiology / Intracellular Signaling Peptides and Proteins - metabolism / Lysosomes / Mammalian cells / Protein transport / Proteins / Signal transduction / SPOPL / Ubiquitin / Ubiquitin-protein ligase / Virus Internalization / Viruses