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EEA1 is a 162kDa membrane bound protein component specific to the early endosome and is essential for fusion between early endocytic vesicles. Early endosomes are cytoplasmic compartments which fuse with endocytic vesicles for redistribution of extracellular compounds to alternate destinations. Zinc-finger-like domains reminiscent of those found in nucleic acid binding proteins are located in the amino and carboxyl-terminal domains of EEA1. The COOH-terminal zinc-finger-like-domain is conserved in several other non-nuclear proteins, some of which are also involved in intracellular protein trafficking. In addition, this domain is an authentic zinc-binding domain and is critical to the intracellular localization of EEA1. Membrane association of EEA1 has been shown to be dependent on phosphatidyl 3-kinase activity, inhibitors of which cause EEA1 to dissociate from early endosomes.
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Regulation of ERα activity by Vps11/18 is independent of intracellular trafficking pathways. a ERα reporter gene assays with HEK293T cells overexpressing Vps11/18 in combination with wild-type (Wt) or K44A mutant (Mut) dynamin II (mean ± s.e.m. with n = 3 biologically independent experiments). b Assays as in a but with knock-downs with the indicated shRNA constructs (mean ± s.e.m. with n = 3 biologically independent experiments). c Transferrin uptake assays in HEK293T cells overexpressing Vps11/18 along with cyan fluorescent protein (CFP). Scale bar indicates 20 µm. d Transferrin uptake assays with immunostaining of EEA1 or LAMP1 in HEK293T cells overexpressing Vps11/18 and blue fluorescent protein (BFP). Scale bar indicates 20 µm. e Assays as in a with knock-downs of HRS or TSG101 (mean ± s.e.m. with n = 3 biologically independent experiments). f–i Assays as in a under the following conditions: treatments to inhibit (Wortmannin, 3-methyladenine (3-MA)) or to stimulate (serum starvation, rapamycin) autophagy (f and g, respectively); knock-down of SQSTM1 (h); treatment with the EGFR inhibitor AG1478 (i) (mean ± s.e.m. with n ≥ 3 biologically independent experiments). j, k Immunoblots displaying PKA substrates and protein levels with or without FI (j) or phospho-ERK1/2 and ERK1/2 protein levels with or without FBS (k) in HEK293T cells overexpressing Vps11/18; FI, cocktail of forskolin and isobutylmethylxanthine to increase intracellular cAMP levels; FBS, fetal bovine serum. l Assays as in a with overexpression of the transcriptional coactivators SRC1 or CARM1 (mean ± s.e.m. with n = 3 biologically independent experiments). Asterisks indicate significant differences with the corresponding negative controls with p-values < 0.05. Statistical significance was determined with unpaired and two-sided Student’s t-tests. Source data are provided as a Source Data file
Image collected and cropped by CiteAb under a CC-BY license from the following publication: Vps11 and Vps18 of Vps-C membrane traffic complexes are E3 ubiquitin ligases and fine-tune signalling. Nat Commun (2019)
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