Monoubiquitination of EEA1 regulates endosome fusion and trafficking
© Ramanathan et al.; licensee BioMed Central Ltd. 2013
Received: 13 March 2013
Accepted: 24 April 2013
Published: 23 May 2013
Early 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.
Here 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.
Our 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.
The survival of eukaryotic cells depends on intake of nutrients from the environment. This process is at least in part dependent on endocytosis, a cellular pathway in which a portion of the plasma membranes invaginate, forming vesicular structures termed endosomes. Endocytosis also imparts regulatory functions in a large number of processes ranging from cellular signaling transduction to protein quality control [1, 2]. Upon entering the cytosol, the early endosomes undergo a complex maturation process as they traverse towards a perinuclear region via the cellular cytoskeletal system. Meanwhile, through acquisition of new proteins and lipids as well as by fusing with other endosome vesicles, early endosomes mature into late endosomes, which eventually fuse with the lysosomes to turn over internalized molecules . It is also known that early endosomes constantly undergo a fission process . Some fission products form recycling endosomes, which help to bring certain internalized membrane receptors back to the plasma membrane for re-usage [5, 6]. In addition to receiving materials from the plasma membranes, the endosomes can also exchange contents with the Golgi apparatus through vesicular trafficking. Thus, the endocytic membrane system is highly dynamic and interactive, a feature required to maintain membrane equilibrium and structural integrity of many intracellular organelles.
Early endosomal autoantigen 1 (EEA1) is an essential component of the endosomal fusion machinery [7, 8]. It contains a C-terminal FYVE domain responsible for binding phosphatidyl inositol-3-phosphate on the membrane, two binding sites that impart communications with the master endocytosis regulator Rab5, and a long helical domain required for oligomerization of EEA1 [7, 9–13]. These features confer membrane binding and fusion-inducing activities to EEA1 because once the membrane-associated EEA1 oligomerizes, it tethers endosomes and primes them for subsequent fusion .
We previously identified EEA1 as a potential substrate for the ubiquitin selective segregase p97 , an ATPase that operates in many cellular processes to separate ubiquitinated polypeptides from membranes, DNA, or large protein complexes [16, 17]. Our results suggested that p97 may regulate endosome fusion by controlling the oligomerization status and therefore the tethering activity of EEA1. In this study, we further demonstrate that similar to other p97 substrates, EEA1 is subject to regulation in cells by ubiquitination, which appears to control not only the size of the endosomes, but also their trafficking pattern.
Monoubiquitination of EEA1 in cells
EEA1 has an intrinsic affinity to ubiquitin conjugating enzymes
Protein ubiquitination usually requires three types of enzymes, an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligase . A conjugation reaction also depends on ubiquitin and ATP. In most cases, these enzymes would form polymerized chains on substrates and the type of ubiquitin chains can dictate the functional consequence of a ubiquitination reaction .
We next wished to reconstitute ubiquitination of EEA1 using purified proteins. We incubated purified EEA1 (Figure 2B), ubiquitin activating enzyme (E1), ubiquitin, and ATP together with a panel of E2 conjugating enzymes. After incubation, immunoblotting showed that several E2 enzymes were able to support EEA1 ubiquitination even in the absence of any E3 ubiquitin ligases. These included Ube2A(HR6a), Ube2H(UbcH2), Ube2D1(UbcH5a) and Ube2E1(UbcH6). Ube2A appeared to be the most efficient one in mediating EEA1 ubiquitination (Figure 2C).
As mentioned above, ubiquitination often involves an E3 ligase that serves as a matchmaker to bring a substrate in proximity to a ubiquitin-charged E2 enzyme . One method that allows bypass of an E3 ligase in ubiquitination is to utilize a ubiquitin recognition motif (UIM) in a substrate, which binds directly to the ubiquitin moiety on an activated E2 [21, 22]. This mechanism requires a hydrophobic surface composed of Ile44 and Leu8 in ubiquitin, which serves as the docking site for UIM. EEA1 does not contain any identifiable UIMs, but it might have a cryptic ubiquitin binding site that mediates its own ubiquitination. To exclude this possibility, we performed in vitro EEA1 ubiquitination using ubiquitin mutants defective in UIM binding . The result showed that these ubiquitin mutants were as effective as wild type ubiquitin in ubiquitination of EEA1 (Figure 2D). This observation raised the possibility that EEA1 may directly communicate with an E2 enzyme(s) to receive ubiquitin from it. This conclusion was indeed confirmed by Surface Plasmon Resonance (SPR) experiments using purified EEA1 and an E2. These in vitro binding experiments demonstrated that EEA1 had an intrinsic affinity to Ube2A with Kd of ~9.7 ± 0.54 μM (Figure 2E, F). By contrast, Ube2G2 bound EEA1 with a significantly reduced affinity (data not shown), consistent with the fact that Ube2G2 could not ubiquitinate EEA1 in vitro.
Expression of a ubiquitin-EEA1 chimera generates giant vacuole-like endosomes
To see if the enlarged endosome phenotype depends on the interplays between Ub-EEA1 and an unknown ubiquitin receptor in cells, we substituted Ile44 in Ub-EEA1 with alanine. This isoleucine residue is required for ubiquitin recognition by all forms of ubiquitin binding domains in cells. Thus, if an ubiquitin binding effector is required for Ub-EEA1 to induce enlarged endosomes, the mutation should abolish the phenotype. Indeed, even though UbI44A-EEA1 was expressed at a higher level than Ub-EEA1, as demonstrated by immunoblotting (Figure 3D), cells expressing this mutant only displayed a small increase in size of endosomes, comparable to cells expressing EEA1 (Figure 3A). Similar observations were made using a set of EEA1 constructs that carried a FLAG tag in addition to the ubiquitin fusion in COS7 cells and in another mammalian cell line, the HEK293 cells (Additional file 1: Figure S2).
EEA1-Ubiquitin expression blocks internalization of transferrin
It was known from previous studies that constitutive activation of Rab5 induces uncontrolled fusion of early endosomes, resulting in giant endosomes . However, endosomes generated by Rab5 activation (e.g. expression of the constitutive activated Rab5) are morphologically distinct from those produced by Ub-EEA1. The former generates enlarged endosomes that are mostly spherical whereas as the endosome sheets formed by Ub-EEA1 are often irregular in shapes. In addition, the endosome structures generated by Ub-EEA1 are also generally larger than those by Rab5 Q79L expression. These differences suggest that uncontrolled fusion may not be the only contributor that forms the abnormal endocytic structures in Ub-EEA1 expressing cells.
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 not only induce endosome fusion, but also 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. This model also explains why the endosomes in Ub-EEA1 cells are even larger than those in Rab5 Q79L expressing cells, as the endosome enlargement induced by Rab5 activation may be solely due to increased vesicle fusion.
We showed previously that the p97 ATPase can regulate the oligomeric state of EEA1 to control endosome morphology and trafficking kinetics . Given that p97 substrates are often polyubiquitinated [27, 28], we investigate whether EEA1 is ubiquitinated in cells. To our surprise, we find that EEA1 preferentially undergoes monoubiquitination in cells. In many p97-mediated biological processes studied to date, the functional consequence of p97 action is to separate ubiquitinated substrates from large immobile structures, therefore facilitating their degradation by the proteasome. This has been best demonstrated for the degradation of misfolded proteins of the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway [29, 30]. Because EEA1 is mostly modified with single ubiquitin moieties, a functional interplay between ubiquitinated EEA1 and p97, if exists, may not be directly involved in p97-dependent modulation of the EEA1 oligomeric state. In support of this notion, we find that unlike many p97 substrates, inhibition of p97 does not significantly increase the levels of ubiquitinated EEA1. In addition, p97 co-precipitates with both modified and unmodified EEA1 with apparently similar affinities (data not shown). Because the activity of the ubiquitin-EEA1 fusion protein is dependent on Ile44 of ubiquitin, it is possible that monoubiquitination of EEA1 may recruit an unknown ubiquitin receptor, which may in turn interfere with p97-dependent disassembly of EEA1. Alternatively, monoubiquitination of EEA1 may compete with a parallel polyubiquitination event essential for disassembly of an EEA1 oligomer complex by p97.
Protein monoubiquitination can occur on many substrates in cells via a variety of mechanisms. One well-known method of monoubiquitination is via an E3 independent mechanism. In this case, monoubiquitination occurs when a substrate make direct contact with a ubiquitin charged E2 enzyme. The substrate can either directly interact with the E2 or use a ubiquitin binding motif to associate with the ubiquitin molecule at the E2 active site . We show that ubiquitination of EEA1 is apparently due to an intrinsic affinity between EEA1 and E2 ubiquitin conjugating enzymes. Our in vitro study suggests that EEA1 can be ubiquitinated by several E2 enzymes in the absence of any E3 ubiquitin ligases. The functional interplay between EEA1 and E2 conjugating enzymes are corroborated by our protein interaction study, which demonstrate a direct interaction between EEA1 and Ube2A. Although Ube2A appears to be the most efficient E2 that ubiquitinates EEA1 in vitro, depletion of Ube2A and Ube2B simultaneously using siRNA-mediated gene silencing does not significantly affect the levels of ubiquitinated EEA1 in cells. It is possible that other E2 enzymes may substitute Ube2A and Ube2B in their absence. Alternatively, Ube2A and Ube2B may not be the major E2 for EEA1 ubiquitination given its preferred nuclear localization. Further experiments would be required to identify the E2s responsible for EEA1 ubiquitination in cells. In addition, our current data cannot exclude that cells may use an E3-dependent mechanism to mono-ubiquitinate EEA1. Despite these uncertainties, the intrinsic affinity between EEA1 and E2 enzymes as demonstrate in this study clearly indicates the likelihood that an EEA1 molecule in cells can just receive ubiquitin directly from one or more E2s, which are abundant in cells.
One unexpected observation of this study is that expression of a Ub-EEA1 fusion protein mimicking ubiquitinated EEA1 induces giant endocytic structures distinct from those generated by over-activating Rab5. Constitutively activated Rab5 recruits wild type EEA1 to the endosome membrane, which results in enlarged endosomes that are sphere-shaped. By contrast, the endosomes generated by Ub-EEA1 expression are larger in size and more irregular in appearance. Interestingly, despite being driven by the same promoter, EEA1 and ubiquitin-EEA1 are expressed in cells at different levels. Ubiquitin moiety fused to EEA1 seems to impart instability, resulting in a much lower level of expression. Ub-EEA1 bound to the endosome membrane may be constantly turned over by the lysosomes that are associated with the abnormal endocytic structures. Although Ub-EEA1 is expressed at a lower level than EEA1, the endosomes generated by Ub-EEA1 are much larger than those formed upon EEA1 expression. Thus, ubiquitinated EEA1 seems to represent an over-activated form that drives the fusion and maturation of those vesicles associated with it. Since the activity of Ub-EEA1 is dependent on isoleucine 44 in the attached ubiquitin, it is reasonable to presume that its activity is dependent on a cellular ubiquitin receptor capable of recognizing both ubiquitin and EEA1. Regulation of the size of intracellular vesicles by monoubiquitination of a fusion factor has not been reported previously. However, a recent study demonstrated that monoubiquitination of the COPII component SEC31 can increase the size of COPII-coated secretory vesicles .
The ubiquitinated EEA1 only constitutes a small fraction of the entire pool of EEA1 at the steady state, in line with other substrates undergoing ubiquitination . This observation suggest that ubiquitinated EEA1 may act catalytically to induce endosome fusion. Moreover, ubiquitination of endogenous EEA1 may be normally antagonized in cells by cellular deubiquitinases, a family of ubiquitin isopeptidases that remove ubiquitin conjugates from substrate proteins . By limiting ubiquitinated EEA1, cells may avoid overflow of endocytic vesicles to the lysosomes. Several deubiquitinases have been found to be associated with the endocytic membrane system including USP8 and AMSH [33–35]. However, siRNA-mediated depletion of these proteins does not affect the levels of ubiquitinated EEA1 in cells (data not shown). Identification of deubiquitinases for EEA1 may further reveal the mechanism and biological significance of EEA1 ubiquitination in the context of endocytic trafficking.
In summary, our study establishes EEA1 as a ubiquitin-regulated factor that may be modified via an E3 independent ubiquitin conjugation mechanism in cells. In addition, ubiquitination may significantly alter EEA1 activity to influence endocytosis.
Cell culture, plasmid preparation, transfection
COS7 and HEK293 cells were purchased from ATCC. Full length human EEA1 was purchased (OriGene). EEA1 deletion mutants were made by introducing SgfI or MluI sites at desired positions using PCR based site-directed mutagenesis. Restriction digestion using MluI or SgfI and religation yielded desired deletion mutants. To make plasmid expressing the EEA1-ubiquitin fusion protein, the ubiquitin open reading frame was amplified with primers carrying mutation to generate a ubiquitin G76V variant. The primers also included EEA1 sequences flanking the ubiquitin sequences. The resulting PCR product was used as primers for subsequent site-directed insertional mutagenesis using EEA1 or EEA1-FLAG expressing plasmid as templates to yield in frame N-terminal Ub G76V fusion to EEA1. Site directed mutagenesis was performed using a kit from Strategen following manufacture’s protocol. TransIT-293 (Mirus) and FuGENE 6 (Roche) were used for plasmid transfection in HEK293 and COS7 cells, respectively.
Antibodies, proteins, and chemicals
Rabbit anti-EEA1 (Cell Signaling), rabbit anti-FLAG (Sigma); mouse anti-EEA1 (BD Biosciences); rabbit anti-β-COP (Thermo Scientific, IL), rabbit PDI (Enzo life sciences, NY), mouse Tom20 (clone F10, Santa Cruz Biotechnology, CA) and mouse Lamp1 (clone H5G11, Santa Cruz Biotechnology, CA) were used. Transferrin-568, goat anti-mouse or goat anti-rabbit conjugated to Alexa-568 and Alexa-488 were from Invitrogen. Goat anti-mouse conjugated to infrared dye IR680 and goat anti-rabbit conjugated to IR800 were from Rockland (Rockville, MD). GST-E1 and ubiquitin conjugating enzyme used in this study were purchased from BostonBiochem. Purification of Ube2g2 was described previously .
Purification of EEA1
To purified EEA1, HEK293 cells were transfected with a plasmid expressing FLAG-tagged full length EEA1. Cells were harvested 72 hours post transfection in a buffer containing 1% DeoxyBig CHAP, 30 mM Tris. HCl pH 7.4, 150 mM potassium acetate, 4 mM magnesium acetate and a protease inhibitor cocktail. The cleared cell extract was incubated with FLAG-agarose beads (Sigma) and the bound materials were extensively washed with a buffer containing 0.1% DeoxyBig CHAP, 30 mM Tris. HCl pH 7.4, 150 mM potassium acetate, 4 mM magnesium acetate. The bound materials were eluted using 0.2 mg/ml 3X FLAG peptide (Sigma) in 25 mM Hepes, pH 7.2, 115 mM potassium acetate, 5 mM sodium acetate, 2.5 mM magnesium acetate.
In vitro ubiquitination
To ubiquitinate EEA1 using PNS, HEK293 cells treated with a hypotonic low salt buffer (50 mM Hepes, 7.3, 10 mM potassium chloride, 2 mM magnesium chloride, 0.5 mM DTT, and a protease inhibitor cocktail) were homogenized with a Dounce-homogenizer. After centrifugation at 1000 g for 5 min at 4°C, the postnuclear supernatant (PNS) fraction was collected and used in an in vitro ubiquitination reaction containing 25 μg HA-tagged ubiquitin, 1 μg ubiquitin aldehyde, 0.2 μg GST-E1, an ATP regenerating system, ~1 mg PNS in a 120 μl volume. After incubation at 37°C, aliquots taken at different time points were lysed in the NP40 lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM sodium chloride, 2 mM magnesium chloride, 0.5% NP40, and a protease inhibitor cocktail). EEA1 immunoprecipitated with FLAG beads was analyzed by immunoblotting. In vitro ubiquitination with purified components were performed in 20 μl reaction buffer (25 mM Tris HCl, pH 7.4, 2 mM magnesium/ATP, and 0.1 mM DTT) containing 40 ng GST-E1, 4 μl HA-ubiquitin, ~0.5 μg EEA1-FLAG, an ATP regenerating system, and 1 μl indicated E2 conjugating enzymes. The reaction was stopped by addition of the Lamili buffer and analyzed by immunoblotting.
Surface plasmon resonance interaction analysis
Surface plasmon resonance interaction experiments were carried out at 25°C on a Biacore 2000 system (GE Healthcare). Purified EEA1-FLAG was covalently attached to a carboxymethyl dextran-coated gold surface (CM5 Chip; GE Healthcare) using an amide couple kit following the manufacturer’s suggested protocol. Briefly, the carboxymethyl group of dextran was activated with N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). EEA1-FLAG in a buffer containing 10 mM sodium acetate (pH 4.5) was injected at a flow rate of 10 μL/min with 2-min contact time, resulting in the immobilization of EEA1-FLAG at a level of ~2,000 RU. Any remaining reactive sites in the flow cell were blocked by ethanolamine. The E2 interactions were monitored at a flow rate of 20 μL/min with the E2 concentration ranging from 2.5 to 160 μM. Analysts were prepared in a buffer containing 10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% (vol/vol) surfactant P20. Regeneration was achieved by injecting a buffer containing 2 M NaCl. SPR signal was normalized by using a reference flow cell containing no EEA1. For each E2 variant, at least 2 measurements were made.
Immunostaining, confocal microscopy and electron microscope analyses
For EEA1 staining, cells plated on coverslips for 24 h were fixed in 2% formaldehyde in PBS for 10 min, rinsed in PBS and blocked for 15 min in PBS containing 10% FBS. Post-blocking, the cells were rinsed and incubated with mouse or rabbit anti-EEA1 primary antibodies (1:100 dilution) in PBS containing 10% FBS and 0.2% saponin. For p97 staining, cells were fixed in 4% paraformaldehyde containing 250 mM sucrose, permeabilized and stained with mouse anti-p97 antibody in PBS containing 0.1% Igepal CA-630 and detected with secondary goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa fluro-568 or 488. To track endocytic cargoes, cells were washed and starved for 30 min in serum-free DMEM containing 0.5% BSA followed by 15 min incubation with labeled Transferrin (5 μg/ml) on ice. The cells were washed twice with PBS and the bound labeled cargoes were allowed to internalize for different time points by incubating the cells at 37°C. All images were obtained using a 510 LSM Confocal microscope (Zeiss) with a 10× Plan Apo objective and processed using Adobe Photoshop 7.0. For electron microscope analyses, COS7 cells were treated with either DMSO or 10 μM EerI for 3 h. Cells were washed with PBS and then fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer at room temperature for 1 h prior to electron microscope analysis.
We thank J. Zimmerberg for helpful discussions, J. Donaldson for Rab5-expressing plasmids. The research is supported by the Intramural Research Program of the NIDDK at the National Institutes of Health.
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