From the
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
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
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).
The
metal-binding finger motifs of EEA1,
Cys-X
The presence of a canonical C
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Marino Zerial for anti-Rab5 and anti-Rab7
antibodies and Jean Gruenberg for helpful comments and suggestions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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.
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).
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 C
H
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 C
H
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.
(
)
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.
-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.
H
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.
/EMBL Data Bank with accession number(s) L40157.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.