©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EEA1, an Early Endosome-Associated Protein
EEA1 IS A CONSERVED -HELICAL PERIPHERAL MEMBRANE PROTEIN FLANKED BY CYSTEINE ``FINGERS'' AND CONTAINS A CALMODULIN-BINDING IQ MOTIF (*)

Fi-Tjen Mu (1), Judy M. Callaghan , Olivia Steele-Mortimer (2), Harald Stenmark (2), Robert G. Parton (2), Paul L. Campbell (3)(§), James McCluskey (3), Jing-Ping Yeo , Edward P.C. Tock (1), Ban-Hock Toh (¶)

From the (1) Department of Pathology, National University of Singapore, Singapore 0511, Singapore, the (2) European Molecular Biology Laboratory, Heidelberg 69021, Federal Republic of Germany, the (3) Centre for Transfusion Medicine and Immunology, Flinders Medical Centre, Adelaide 5042, South Australia, and the Department of Pathology and Immunology, Monash Medical School, Melbourne 3181, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly -helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine ``finger'' motifs. The COOH-terminal fingers, Cys-X-Cys-X-Cys-X-Cys and Cys-X-Cys-X-Cys-X-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.


INTRODUCTION

Endocytosis is a process whereby cells internalize extracellular molecules into cytoplasmic vesicles (Courtoy, 1991). In receptor-mediated endocytosis, ligands bind receptors on clathrin-coated pits, which invaginate forming coated vesicles; the vesicles uncoat and transport receptor-ligand complexes to early endosomes (Trowbridge et al., 1993). Early endosomes are a major sorting compartment from which ligands may be released and transported to lysosomes and receptors recycled to the plasma membrane, or from which ligand-receptor complexes are transported to lysosomes, to the opposite side of polarized cells, or recycled to the plasma membrane (Shepherd, 1989). Ligands, dissociated by low endosomal pH, may be confined to the main body of endosomes and follow a nonselective default pathway to late endosomes and lysosomes (Mellman, 1993). Recycling receptors may escape the degradative pathway via tubular extensions and be sorted by specific interaction of their cytoplasmic tails with peripheral membrane proteins for targeting to distinct locations. Two models have been proposed for transport from early to late endosomes. The ``vesicle shuttle model'' proposes that early and late endosomes are preexisting compartments communicating via microtubule-dependent vesicular traffic (Griffiths and Gruenberg, 1991). The ``maturation model'' suggests that early endosomes gradually ``mature'' into late endosomes and lysosomes (Murphy, 1991).

Very little is known of proteins associated with early endosomes. The best characterized compartment-specific proteins are the small GTP-binding rab proteins with Rab5 localizing to early endosomes and Rab7 to late endosomes (Chavrier et al., 1990; Gould, 1992). Here we report the characterization and cloning of human EEA1, a hydrophilic 180-kDa peripheral membrane protein on early endosomes. EEA1 shares 17-20% homology with the myosins and contains a calmodulin-binding IQ motif associated with these proteins. EEA1 is a predominantly -helical protein flanked by NH-terminal and COOH-terminal cysteine-rich, metal-binding ``finger'' motifs. The COOH-terminal finger motifs are highly conserved with a peptide encoded by the ZK632 cosmid from Caenorhabditis elegans and by the FAB1 gene of Saccharomyces cerevisiae. The latter has been described as a gene required for the endocytic-vacuolar pathway and nuclear migration. Similar finger motifs, Cys-X-Cys-X-Cys-X-Cys and Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys, are present, respectively, in S. cerevisiae VAC1 (Weisman and Wickner, 1992) and Vps18p (Robinson et al., 1991) proteins, both of which are required for vacuolar sorting. We propose that EEA1 has a role in early endosomal transport and that its conserved cysteine-rich, metal-binding fingers have a role in this process.


MATERIALS AND METHODS

Antibodies

The autoimmune serum was from a patient with subacute cutaneous systemic lupus erythematosus identified in the Monash Clinical Immunology Laboratory. Rabbit antibody to EEA1 was raised against a bacterial fusion protein incorporating the carboxyl terminus of the protein (see below). Mouse anti-Rab5 monoclonal antibodies (Chavrier et al., 1990) and affinity-purified rabbit anti-Rab7 antibody were from Marino Zerial (EMBL).

Immunofluorescence

HeLa and 3T3 cells were fixed in 2-2.5% paraformaldehyde/PBS() for 10-20 min and permeabilized with 0.5% saponin (Sigma)/PBS for 10 min or 1% Nonidet P-40/PBS for 5 min. Fixed cells or Hep-2 cells (Kallestad), were reacted with the autoimmune serum diluted 1:200 in PBS, 0.5% saponin or with affinity-purified human or rabbit antibodies to EEA1. Cells were washed 3 times with PBS, 0.5% saponin and incubated with fluorescein isothiocyanate-conjugated sheep anti-human Ig (Wellcome).

Dual Immunofluorescence Confocal Microscopy

HeLa cells were fixed with 3% paraformaldehyde/PBS for 15 min; free aldehyde groups were quenched with 50 mM NHCl/PBS; and cells were permeabilized with 0.1% Triton X-100/PBS for 4 min. The cells were reacted with anti-transferrin receptor antibody (mouse monoclonal B3-25, Boehringer Mannheim, Germany) at 1:100 dilution and the affinity-purified human autoimmune serum. After three washes in PBS, anti-transferrin receptor was visualized with rhodamine-labeled donkey anti-mouse antibody (DiaNova, Germany) at a 1:100 dilution, and EEA1 was visualized with fluorescein isothiocyanate-labeled goat anti-human Ig. Dual immunofluorescence confocal microscopy was also carried out with a Rab5-overexpressing HeLa clone, 2-1-6-1 (Stenmark et al., 1994a, 1994b). Cells were reacted with mouse anti-Rab5 monoclonal antibody (1:20 dilution in 5% bovine serum), affinity-purified rabbit anti-Rab7 antibody (1:8 dilution), or the affinity-purified autoimmune serum. The cells were incubated with fluorescein isothiocyanate-labeled goat anti-human Ig, rhodamine-labeled donkey anti-mouse Ig (DiaNova), or anti-rabbit Ig (DiaNova).

Immunoelectron Microscopy

Endocytic compartments of HeLa cells were labeled with endocytic tracers (5 nm of BSA-gold for the early endosomes and 16 nm of BSA-gold for late endosomes and lysosomes) as described previously (Chavrier et al., 1990) except that 5 nm of BSA-gold (A = 30) was internalized for 10 min at 37 °C. The cells were then fixed with 8% paraformaldehyde in 250 mM HEPES (pH 7.35), processed for cryosectioning, and sectioned, and the thawed sections were labeled with affinity-purified anti-EEA1 antibody followed by protein A-gold (Chavrier et al., 1990; Griffiths et al., 1984). After embedding in methylcellulose/uranyl acetate, the sections were viewed at an accelerating voltage of 80 kV in a Zeiss transmission electron microscope.

Subcellular Fractionation

HeLa or 3T3 cells were washed 3 times with PBS, detached with a rubber policeman, and resuspended in hypotonic buffer (0.01 M NaCl, 0.003 M MgCl, 0.01 M Tris (pH 8.5) containing phenylmethylsulfonyl fluoride (1 mM), leupeptin (0.5 µg/ml), pepstatin (1.0 µg/ml), and aprotinin (60 µg/ml)) on ice for 10 min. Cells were lysed with a 21-gauge needle and sequentially centrifuged at 600 g to pellet nuclei, 10,000 g to pellet mitochondria, and 100,000 g to pellet membranes. The supernatant after the last centrifugation was kept as the cytosolic fraction. The pellets, resuspended in 50 mM Hepes buffer (pH 7.6) containing 1 mM EDTA and the cytosolic fraction were stored at -70 °C. Protein content of fractions was determined using the BCA protein assay (Pierce).

Immunoblotting

Subcellular fractions of HeLa or 3T3 cells, aqueous and detergent phases of cells partitioned with Triton X-114 (Kooy et al., 1992), membrane fractions extracted with 0.3 M NaCl for 30 min on ice, and affinity-purified bacterial fusion protein were separated by 7.5 or 10% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose and immunoblotted with affinity-purified human and rabbit antibodies to EEA1 as described previously (Yeo et al., 1994). Immunoblots were also carried out with total HeLa cells and chicken fibroblasts under reducing and nonreducing conditions. Total HeLa cell lysates (100 µg) were processed for two-dimensional nonequilibrium pH gel electrophoresis/SDS-PAGE and immunoblotting as described (Mu et al., 1988).

Molecular Cloning

5 10 plaque-forming units of gt11 Hela cell cDNA expression library (Clontech) were screened with the autoimmune serum, diluted 1:1000 in PBS, 3% casein as described previously (Yeo et al., 1994). Positive plaques, identified with I-labeled protein A (2 10 cpm/ml), were purified by 3 cycles of immunoscreening. cDNA inserts were subcloned into pBluescribe (Stratagene), for double-stranded DNA sequencing by the dideoxy chain termination method. To obtain the full-length clone, a human hepatoma cDNA library (Stratagene) was screened with P-labeled DNA probes. Positive plaques were purified and plasmid rescued according to the manufacturer's instructions. Analyses were carried out using the MacVector program (International Biotechnologies, Inc.) or using software provided by the Australian National Genome Information Service. The cDNA sequence was compared with GenBank and EMBL data bases using the FASTA program (Lipman and Pearson, 1985). The translated amino acid sequence was compared with the translated GenBank data base as well as with the Swiss-Prot protein data base. The Swiss-Prot data base was also searched using the pattern library search program of Smith and Smith(1990).

Nucleotide Blot Analysis

For DNA blots, human leucocyte genomic DNA (12 µg) was digested with restriction enzymes, subjected to electrophoresis in 0.6% agarose gel, and transferred to nitrocellulose. Filters were prehybridized for 4 h with 5 saline/sodium phosphate/EDTA, 0.5% SDS, 0.5% skim milk, 1% SDS, 10% dextran sulfate, and 0.5% mg/ml herring sperm DNA; hybridized with P-labeled probe (specific activity >5 10 cpm/µg DNA) overnight at 65 °C; washed with 1 SSC, 0.5% SDS at 65 °C for 30-45 min; and autoradiographed. For RNA blots, total RNA isolated from HeLa cells was enriched for poly(A) RNA by oligo(dT)-cellulose chromatography. Poly(A) RNA was fractionated on 1% agarose formaldehyde-denaturing gel, transferred to nitrocellulose, and probed with P-labeled DNA as described for DNA blots. Multiple tissue RNA blots (Clontech) were also probed.

Production of Antibody to Recombinant Fusion Protein

pFM1, containing 900 bp of coding sequence encoding the carboxyl terminus of EEA1, was subcloned into pGEX 2T (Pharmacia Biotech Inc.) vector. Escherichia coli were transformed with pGEX 2T or pGEX 2T containing pFM1 insert. The bacteria were induced to produce glutathione S-transferase or bacterial fusion protein with 1 mM isopropyl-1-thio--D-galactopyranoside (Kooy et al., 1992). Bacteria were suspended in PBS, 1% Triton X-100, sonicated, and centrifuged at 10,000 g. Soluble glutathione S-transferase or fusion protein in the supernatant was purified by glutathione-agarose chromatography. Rabbits were immunized with 50 µg of purified fusion protein in Freund's complete adjuvent, boosted after 4 weeks with 50 µg of protein in incomplete adjuvent and bled after 2 weeks. Rabbit sera were tested by immunofluorescence and immunoblotting of 3T3 cells.

Affinity Purification of Human and Rabbit Antibodies

1 mg of purified glutathione S-transferase or bacterial fusion protein were bound to 1 ml of swollen CNBr-activated Sepharose 4B beads (Pharmacia). Human autoimmune and rabbit immune sera were diluted 1:4 in PBS and recycled 3 times through the fusion protein column. In addition, the rabbit antibody was also recycled 3 times through a glutathione S-transferase column. The columns were washed extensively with PBS. The run-through from both columns was collected. Bound antibody, eluted with 3 M KSCN, was dialyzed against PBS and concentrated with a microconcentrator (Amicon).


RESULTS

EEA1 Co-localizes with Transferrin Receptor and with Rab5 but Not with Rab7

The autoimmune serum reacted by immunofluorescence with multiple small vesicles distributed throughout the cytoplasm of human Hep-2 and HeLa cells, mouse 3T3 cells, and chicken fibroblasts (data not shown). These results and the immunoblotting data (see below) indicate that EEA1 is evolutionarily conserved. Affinity-purified human autoantibody to EEA1 and affinity-purified rabbit antibody to a bacterial fusion protein incorporating the carboxyl terminus of EEA1 gave the same immunofluorescence reactions (data not shown). Since the cytoplasmic vesicles reactive with the affinity-purified anti-EEA1 antibodies were reminiscent of endocytic structures, we carried out experiments to identify them using dual immunofluorescence confocal microscopy and endocytic markers. In HeLa cells, EEA1 co-localized extensively with the transferrin receptor (Fig. 1, A and A`), an established marker of early endosomes (Hopkins, 1983; Schmid et al., 1988). Since the image shown is a 0.5-µm optical section, plasma membrane-associated transferrin receptors were not readily detected. Localization of EEA1 to early endosomes was supported by co-localization with Rab5, a GTPase specifically localized to early endosomes (Chavrier et al., 1990), in HeLa cells moderately overexpressing Rab5 (Fig. 1, B and B`), and in Madin-Darby canine kidney cells (not shown). In contrast, localization of EEA1 showed little overlap with that of Rab7 (Fig. 1, C and C`), a GTPase found on late endosomes (Chavrier et al., 1990), indicating that EEA1 is not associated with the whole endocytic pathway.


Figure 1: Co-localization of EEA1 with transferrin receptor and Rab5 (arrowheads) but not with Rab7 using confocal dual immunofluorescence microscopy. HeLa cells (panelsA and A`) or Rab5-overexpressing HeLa cell clone 2-1-6-1 (panelsB, B`, C, and C`) were fixed and permeabilized (see ``Materials and Methods''). Cells were reacted with affinity-purified human anti-EEA1 antibody (A-C) and either with mouse monoclonal antibody to transferrin receptor (A`) or Rab5 (B`), or with affinity-purified rabbit anti-Rab7 antibody (C`). Bound antibody was traced with fluorescein isothiocyanate-labeled anti-human Ig (A-C) or with rhodamine-labeled anti-mouse (A` and B`) or anti-rabbit (C`) Ig.



EEA1 Is Associated with Tubulovesicular Early Endosomes Containing Internalized BSA-Gold

The intracellular distribution of EEA1 was examined at the ultrastructural level by immunolabeling ultrathin sections of HeLa cells, which had internalized endocytic markers. Labeling for EEA1 was predominantly associated with tubulovesicular early endosomes containing BSA-gold internalized as a fluid phase marker for 10 min at 37 °C (Fig. 2). Labeling was rarely associated with late endosomes, Golgi cisternae (results not shown), and the plasma membrane (for example, see Fig. 2A). In some cases, significant labeling was observed in close proximity to early endosomes but some distance away from the endosomal membrane (for example, see Fig. 2C). Whether this simply reflects labeling of the cytoplasmic surface of peripheral endosomal elements, which are not clearly visible in the plane of the section or of other endosome-associated material, is presently unclear.


Figure 2: Immunoelectron microscopic localization of EEA1 in HeLa cells. HeLa cells were incubated with 5 nm of BSA-gold to label early endosomes and 16 nm of BSA-gold to label late endosomes and lysosomes (see ``Materials and Methods''). After fixation, thawed cryosections were labeled with affinity-purified antibody to EEA1 followed by 10 nm of protein A-gold. PanelA shows a representative image of the cell periphery. Labeling for EEA1 (10 nm of gold, arrowheads) co-localizes with the internalized 5 nm BSA-gold (smallarrows). Note that there is negligible labeling for EEA1 in the cytoplasm and on mitochondria (m) and generally low labeling associated with the plasma membrane (pm). PanelsB and C show higher magnification views of early endosomes. The labeling for EEA1 (10 nm of gold) is associated with the tubulovesicular early endosomes (labeled with 5 nm of BSA-gold). In panelC, three of the gold particles (arrowheads) are clearly associated with the cytoplasmic surface of the early endosome. EEA1 labeling was rarely observed on late endosomes, labeled with 16 nm of gold (results not shown). Bars, 100 nm.



EEA1 Is a 180-kDa Peripheral Membrane Protein

The autoimmune serum and affinity-purified antibody to EEA1 immunoblotted a 180-kDa protein in HeLa cells, 3T3 cells, and chicken fibroblasts under both reducing and nonreducing conditions (data not shown). The 180-kDa protein was detected in membrane and cytosolic subcellular fractions of HeLa or 3T3 cells (Fig. 3), exclusively in the aqueous phase of cell extracts after Triton X-114 solubilization (Fig. 4) and was extracted from membranes by 0.3 M NaCl (data not shown). These observations suggest that EEA1 is a peripheral membrane protein. The results are consistent with the prediction that EEA1 is a hydrophilic protein lacking hydrophobic transmembrane domains (see below). Immunoblots with membrane fractions were developed by enhanced chemiluminescence (Fig. 3) as the signal developed by 4-choloro-1-napthol was very weak. These results suggest that either there is a larger pool of EEA1 in the cytosol than those associated with membranes or that EEA1 is easily dissociated from membranes during fractionation. Two-dimensional immunoblots showed that EEA1 has an acidic pI (data not shown), consistent with the predicted pI of the protein (see below).


Figure 3: Immunoblotting of subcellular fractions of 3T3 cells with affinity-purified human or rabbit antibody to EEA1. Nuclear, mitochondria, and cytosol fractions (10 µg/lane), or membranes (50 µg/lane) were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with affinity-purified human (A) or rabbit (C) anti-EEA1 antibody or with normal human (B) or rabbit (D) serum. Bound antibody was detected with anti-human or anti-rabbit Ig and enhanced chemiluminescence for nuclear mitochondria, and membrane fractions or 4-choloro-1-napthol/H0 for cytosol fraction.




Figure 4: Immunoblot of Triton X-114 fractions of 3T3 cells with affinity-purified anti-EEA1 antibodies. 3T3 cells were extracted with 0.5% Triton X-114. Aqueous and detergent fractions were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with affinity-purified human (Aff. pur. EEA1 Ab) or rabbit (Aff. pur. rabbit Ab) anti-EEA1 antibody or with normal human (NHS) or rabbit (NRS) serum. Bound antibody was detected as described in the legend to Fig. 3.



Cloning of cDNA Encoding EEA1

A single positive plaque (pFM1) containing a 1.2-kilobase insert was isolated after screening 5 10 plaque-forming units of a HeLa cell expression library with the autoimmune serum (Fig. 5). Two positive clones (pFM5.5 and pFM3.5) were obtained after screening a human hepatoma zap cDNA library with P-labeled pFM1. A further clone (pFM9.9) was obtained from the same library after probing the library with a 500-bp HindIII fragment of pFM3.5. All four clones are probably derived from the same transcript since all overlapping regions had identical sequences. Four individual human genomic DNAs, digested with EcoRI, BamHI, and HindIII and probed with P-labeled pFM1 gave a single fragment after EcoRI and BamHI digestion and two fragments after HindIII digestion (data not shown), consistent with an internal HindIII site in the cDNA. P-labeled pFM1 hybridizes with a 9-kilobase mRNA in a poly(A) RNA preparation from HeLa cells (data not shown). A similar sized message was found in a multiple tissue blot of skeletal muscle, heart, brain, lung, liver, and pancreas (data not shown). To exclude concatamer formation during library construction, pFM9.9, the cDNA clone containing the internal EcoRI site, was digested with EcoRI, and the fragment 5` of this restriction enzyme site was hybridized to HeLa poly(A) RNA or to human multiple tissue blot. The same 9-kilobase message was obtained (data not shown), indicating that the 5` fragment is a related fragment.


Figure 5: Restriction map of full-length and partial length EEA1 cDNA clones. B, BamHI; Eg, EagI; P, Pst; H, HindIII; E, ecoR1;X, Xho.



Sequence Analysis of EEA1

The nucleotide sequence of pNUSPath-2 derived from the four overlapping cDNA clones comprises 4962 bp with a coding region of 4233 nucleotides (Fig. 6). The DNA sequence of EEA1 cDNA is virtually identical with that of locus HSP162 (Homosapiens p162), deposited in the GenBank data base on April 26, 1994 by H. P. Seelig (accession X78998). The coding region encodes a protein of 1411 amino acids with a predicted molecular mass of 162.46 kDa and pI of 5.38. Plots of hydrophilicity (Kyte and Doolittle, 1982), surface probability (Janin et al., 1978; Emini et al., 1985), flexibility (Karplus and Schulz, 1985), antigenic index (Jameson and Wolf, 1988), and amphiphilicity (Eisenberg et al, 1984a, 1984b; von Heijne, 1986) predict a largely hydrophilic structure with short amphiphilic regions in the 15 amino-terminal amino acids and in segments centered around amino acids 515 and 645. Secondary structure predictions (Chou and Fasman, 1974a, 1974b, 1978a, 1978b; Robson and Suzuki, 1976; Garnier et al., 1978) suggest a predominantly -helical structure throughout the full length of the protein with the exception of the extreme NH- and COOH-terminal segments, which contain a number of proline residues and are predicted to have a high content of turns and -sheet. It is notable that these regions contain potential metal-binding motifs (see below).


Figure 6: cDNA and predicted amino acid sequence of EEA1. NH-terminal CH zinc finger motif and COOH-terminal cysteine-rich metal-binding fingers are boxed. IQ motif is underlined. Segment sharing sequence similarity with S. cerevisiae FAB1 and C. elegans CEZK632 proteins is in boldface.



The DNA sequence of the EEA1 cDNA from positions 505 to 4095 shares 52.4% homology with a region of the Entamoeba histolytica myosin heavy chain gene (ENHMHCAX locus) extending from position 2921 to 6512. Optimal alignments of translated amino acid sequence corresponding to this region (EEA1 amino acids 154-1322) (Needleman and Wunch, 1970; Smith and Waterman, 1981; Rechid et al., 1989) using a gap weight of 3.0 and a length weight of 0.1 gives an amino acid identity of 24.5% with 46.7% similarity when conservative substitution are accounted for. Indeed, the translated amino acid sequence of EEA1 has a low but statistically significant global homology with a large number of vertebrate and nonvertebrate myosin heavy chains from both muscle and non-muscle, typically showing amino acid identity of 17-20% across the whole protein with an additional large number of conservative substitutions. However, there is no evidence for a globular myosin ``head'' region in EEA1. Significant overall amino acid identity of 17.9% was also found with the human kinetochore protein CENP-E (Yen et al., 1992). Homology to these proteins is scattered throughout the EEA1 polypeptide, and no large region shows greater identity overall. Statistical evaluation revealed that the homology is unlikely to be due to chance alone, since random shuffling of the myosin or CENP-E sequences generated much lesser degrees of similarity. EEA1 also contains close to its COOH terminus a motif related to the IQ motif and found in the neck regions of all myosins and in the calmodulin binding domains of non-myosin proteins. Alignment of these IQ motifs in EEA1, neuromodulin and in the myosins is shown in Fig. 7. These motifs may be involved in binding of the calmodulin/EF-hand superfamily of proteins, which includes the essential and regulatory light chains of conventional myosins (Cheney and Mooseker, 1992).


Figure 7: Potential calmodulin-binding IQ motifs of EEA1, neuromodulins, and the unconventional myosins. Boxes indicate amino acid identity or conservative substitution in at least two of the aligned proteins.



A short region of 57 amino acids at the extreme COOH terminus showed 49.1% identity with a segment of the CEZK632 locus in the GenBank data base. The sequence of this locus originated in cosmid ZK632 containing C. elegans sequence, which was sequenced as part of the C. elegans sequencing project (Sulstan et al., 1992) and has not been attributed with any function. However, the implied product of the FAB1 gene, which is required for endocytic-vacuolar pathway and nuclear migration in S. cerevisiae (locus U01017, submitted to GenBank by A. Yamamoto and D. Koshland, 1993) also shows a striking homology with this region of EEA1, despite little homology being present elsewhere in the protein. Alignment of the corresponding regions from EEA1, CEZK632, and FAB1 is shown in Fig. 8. This region spans a potential metal binding finger present in EEA1 as Cys-X-Cys-X-Cys-X-Cys and Cys-X-Cys-X-Cys-X-Cys. It is notable that the cysteines comprising this element are absolutely conserved across the three implied proteins. EEA1 also contains a canonical zinc-finger motif of the CH type (Klug and Rhodes, 1987; Evans and Hollenberg, 1988; Payre and Vincent, 1988; Miller et al., 1985; Berg, 1988) close to the NH terminus ( Fig. 6and Fig. 8).


Figure 8: Homology of carboxyl-terminal cysteine finger motifs of EEA1 with those of C. elegans ZK632 and S. cerevisiae FAB 1 proteins. Boxes indicate amino acid identities or conservative changes between EEA1 and at least one of the other two proteins.



The EEA1 protein also contains multiple consensus sequences for N-glycosylation, casein kinase II phosphorylation, N-myristoylation, protein kinase C phosphorylation, tyrosine kinase phosphorylation, and for the leucine zipper structure in addition to single consensus motifs for glycosaminoglycan attachment and cAMP- and cGMP-dependent protein kinase phosphorylation. Due to a lack of specificity in these consensus motifs, the significance of this analysis is not clear, although post-translational modification of EEA1 may contribute to the difference between its observed and predicted molecular weight.

Rabbit Antiserum to Recombinant Protein Reacts with Cytoplasmic Vesicles and with a 180-kDa Molecule

The 1.2-kilobase EcoRI cDNA insert (pFM1) containing a 900-bp coding region was subcloned into pGEX, a vector that expresses a bacterial fusion protein at the carboxyl terminus of glutathione S-transferase. The size of the 60-kDa fusion protein (Fig. 9) obtained by induction with isopropyl-1-thio--D-galactopyranoside was in good agreement with the predicted combined size of glutathione S-transferase (27 kDa) and that of the partial polypeptide encoded by the cDNA clone (33 kDa). A rabbit antiserum against the fusion protein, and affinity-purified rabbit anti-fusion protein antibody, reacted with cytoplasmic vesicles and immunoblotted the fusion protein (Fig. 9B) and a 180-kDa 3T3 cell protein ( Fig. 3and Fig. 4). The affinity-purified rabbit antibody reacted with glutathione S-transferase but after purification on a glutathione S-transferase affinity column all reactivity was abolished (Fig. 9C). The affinity-purified autoantibody also reacted with the fusion protein (Fig. 9B), but it did not react with glutathione S-transferase (Fig. 9C). The human autoimmune serum was recycled through the glutathione S-transferase affinity column using the same conditions as those used with the fusion protein affinity column. Antibody titer remained unchanged after recycling through this column.


Figure 9: Immunoblotting of bacterial fusion protein with affinity-purified human or rabbit antibody to EEA1. pFM1 containing 900 bp of coding sequence was subcloned into pGex 2T expression vector. Fusion protein induced by isopropyl-1-thio--D-galactopyranoside was purified on glutathione affinity column. PanelA, total proteins from noninduced wild-type pGex 2T, pGex 2T subcloned with pFM1, affinity purified pGex-pFM1, pGEX-GST, and affinity purified pGex-GST were electrophoresed on 10% SDS-PAGE and stained with Coomassie Blue. PanelB, immunoblots of fusion protein with affinity-purified human (Aff. pur. EEA1 Ab) or rabbit (Aff. pur. rabbit Ab) antibody to EEA1, or with normal human (NHS) or rabbit (NRS) serum. PanelC, immunoblots of glutathione S-transferase protein with rabbit anti-glutathione S-transferase antibody, or with affinity-purified human or rabbit antibody to EEA1. Bound antibody was detected as described in legend to Fig. 3.




DISCUSSION

We have cloned the cDNA encoding a 180-kDa protein, named EEA1. EEA1 is evolutionarily conserved as it is present in human, mouse, and chicken cells. It associates with early endosomes since it co-localized with the transferrin receptor and Rab5 and not with Rab7. Localization to early endosomes was confirmed by ultrastructural studies showing co-localization of EEA1 with tubulovesicular early endosomes containing internalized BSA-gold. EEA1 is a hydrophilic peripheral membrane protein lacking a hydrophobic transmembrane domain. It is present in cytosolic and in membrane fractions, it fractionates exclusively in the aqueous phase after Triton X-114 solubilization, and it is extracted from membranes by 0.3 M NaCl. These properties are different from those of a 195-kDa integral membrane protein associated with early endosomes (Pitt and Schwartz, 1991). CLIP-170 is an early endosomal protein that links early endocytic vesicles to cytoplasmic microtubules by a novel motif present in a tandem repeat in the amino-terminal domain (Pierre et al., 1992). Like EEA1, it has a cysteine finger motif at the carboxyl terminus. However, the finger motifs and the amino acid sequence of EEA1 are different from those of CLIP-170, and EEA1 lacks the microtubule binding motif of CLIP-170.

The sequence of EEA1 gene is virtually identical to that of HSP 162, a cDNA derived from HeLa cells (GenBank accession number X78998).() Indeed the sequence differences between EEA1 and HSP 162 are limited to a dinucleotide substitution giving rise to the replacement of Ala by Ser at amino-acid 277 in EEA1, as well as a silent single base substitution at nucleotide 245 in EEA1. In addition, a three-nucleotide deletion is present in the 5` noncoding region of EEA1 as well as a two-nucleotide deletion followed by a dinucleotide substitution in the 3` noncoding region.

The metal-binding finger motifs of EEA1, Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys, are strikingly conserved within a homologous segment of a protein encoded by the ZK632 cosmid of C. elegans and the S. cerevisiae FAB1 protein. The latter has been implicated in the endocytic-vacuolar pathway and in nuclear migration. The presence of this motif in EEA1 and FAB1, which although globally dissimilar in sequence and yet are both implicated in endocytic trafficking, suggests a common function for this region of both proteins. These observations also suggest a possible related role for the homologous protein encoded by the C. elegans ZK632 cosmid. Similar finger motifs are present as Cys-X-Cys-X-Cys-X-Cys in the S. cerevisiae VAC1 gene, which is required for vacuole inheritance and vacuole protein sorting (Weisman and Wickner, 1992) and as Cys-X-Cys-X-Cys-X-Cys in the 190-kDa bovine microtubule-associated protein-U (Aizawa et al., 1990). A cysteine-rich finger motif, Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys-X-Cys, at the carboxyl terminus of S. cerevisiae Vps18p protein has been localized to the cytosolic face of vacuoles (Robinson et al., 1991). A mutant in which the first cysteine of this motif was changed to serine resulted in a temperature-conditional sorting defect. Another yeast protein, Vps11, which also has a cysteine-rich motif near the carboxyl terminus, similar to Vps18p, co-localizes with Vps18p (Preston et al., 1991). These observations suggest that the finger motifs are necessary for vacuolar protein sorting in yeast. On the basis of these observations, we propose that the two metal-binding cysteine finger motifs of EEA1 are required for early endosomal sorting.

The presence of a canonical CH type zinc finger at the amino terminus of EEA1 is of unknown significance since this motif has usually been found in nucleic acid binding proteins. However, there are some 40 other proteins in the SWISS-PROT data base that are identified by this motif, and a metal binding function cannot be excluded in these cases or in the case of EEA1.

EEA1 shares 17-20% sequence identity with both conventional and unconventional myosins and contains a potential calmodulin binding IQ motif typically found in the neck region of unconventional myosins (Cheney and Mooseker, 1992). Unconventional myosins have been implicated in vesicular transport (Bretscher, 1993; Titus, 1993). However, EEA1 lacks the globular ATP and actin binding head domain to qualify it as an ``unconventional'' myosin. Nonetheless, it seems likely that EEA1 shares an evolutionary relationship with the unconventional myosins given its homology with these proteins and the presence of the calmodulin-binding IQ motif.

The IQ motif in EEA1 suggests that calmodulin may be implicated in the function of EEA1. However, EEA1 lacks the sequence ASF, which follows the IQ motif of neuromodulin, a 24-kDa calmodulin-binding non-myosin protein (Chapman et al., 1991). The phenylalanine associates with calmodulin via a hydrophobic interaction, which is disrupted following protein kinase C-dependent phosphorylation of the adjacent serine, which introduces a strong negative charge. Thus calmodulin binds to neuromodulin in the absence of calcium and is released following calcium-dependent protein kinase C phosphorylation. Similar hydrophobic residues are present in many (although not all) of the unconventional myosins.

The function of EEA1 remains to be determined. Based on its localization to early endosomes and the similarity of its carboxyl-terminal zinc fingers to proteins regulating membrane traffic, we propose as a working hypothesis that EEA1 has a role in vesicular transport of proteins through early endosomes. The conserved metal-binding cysteine finger motifs may be crucial in this process.


FOOTNOTES

*
This study was supported in part by grants from the National University of Singapore, the Mary Whight Lupus Fellowship and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L40157.

§
Supported by a National Health and Medical Research Council Australian Postdoctoral Fellowship.

To whom correspondence should be addressed: Dept. of Pathology and Immunology, Monash Medical School, Commercial Rd., Prahran, Victoria 3181, Australia. Tel.: 613-276-2713; Fax: 613-529-6484.

The abbreviations used are: PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

H. P. Seelig, unpublished results.


ACKNOWLEDGEMENTS

We thank Marino Zerial for anti-Rab5 and anti-Rab7 antibodies and Jean Gruenberg for helpful comments and suggestions.


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