Abstract
BackgroundEarly endosomal autoantigen 1 (EEA1) is a membrane tethering factor required for the fusion and maturation of early endosomes in endocytosis. How the activity of EEA1 is regulated in cells is unclear.ResultsHere we show that endogenous EEA1 is prone to monoubiquitination at multiple sites, owing to an intrinsic affinity to ubiquitin conjugating enzymes (E2). The E2 interactions enable a ubiquitin ligase (E3) independent mechanism that decorate EEA1 with multiple mono-ubiquitin moieties. Expression of an ubiquitin-EEA1 chimera that mimics native mono-ubiquitinated EEA1 generates giant endosomes abutting the nucleus. Several lines of evidence suggest that this phenotype is due to increased endosome fusion and a simultaneous blockade on an endosome recycling pathway. The latter is likely caused by diminished endosome fission in cells expressing ubiquitin-EEA1.ConclusionOur results demonstrate that ubiquitination may dramatically affect the activity of an endosome fusion factor to alter endosome morphology and trafficking pattern, and thereby implicating an unexpected role of ubiquitin signaling in endocytosis.
Highlights
Endosomal autoantigen 1 (EEA1) is a membrane tethering factor required for the fusion and maturation of early endosomes in endocytosis
Monoubiquitination of Early endosomal autoantigen 1 (EEA1) in cells To investigate whether EEA1 is ubiquitinated in cells, we expressed full-length FLAG-tagged wild type EEA1 together with hemagglutinin (HA)-tagged ubiquitin in HEK293 cells
These additional EEA1containing species corresponded to ubiquitinated EEA1 because they were recognized by anti-HA antibodies, as demonstrated by two-color immunoblotting (Figure 1A)
Summary
Monoubiquitination of EEA1 in cells To investigate whether EEA1 is ubiquitinated in cells, we expressed full-length FLAG-tagged wild type EEA1 together with hemagglutinin (HA)-tagged ubiquitin in HEK293 cells. Immunostaining with a LAMP1 antibody that labels lysosomes showed that many lysosome vesicles were docked on the large endocytic structures formed upon Ub-EEA1 expression (Figure 4J-L) These results suggest that Ub-EEA1 disrupts endocytic homeostasis at least in part by inducing uncontrolled fusion of endosomes that can fuse further with the lysosomes. Ubiquitin-EEA1 traps a cell surface receptor at endosomes One possible explanation for the enlarged endosome phenotype associated with Ub-EEA1 is that activation of EEA1 by ubiquitination may induce endosome fusion, and block an endosome fission process required for recycling of certain membrane receptors such as the transferrin receptor to the plasma membrane This could lead to a depletion of transferrin receptor from the cell surface, resulting in abnormal transferrin uptake.
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