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From the Program in Molecular Medicine and Departments of
§ Physiology and
Received for publication, September 25, 2001
Early endosome antigen 1 (EEA1) is a 170-kDa
polypeptide required for endosome fusion in mammalian cells. The COOH
terminus of EEA1 contains a FYVE domain that interacts
specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a
Rab5 GTPase binding region adjacent to the FYVE domain. The dual
interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized
to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids
1277-1411) and full-length EEA1 constructs containing point mutations
in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These
constructs localized to endosomes in intact cells as efficiently as
their wild-type counterparts. Furthermore, overexpression of the
truncated constructs, both wild-type and mutated, impaired the function
of endogenous EEA1 resulting in the accumulation of small, untethered
endosomes. These results suggest that association with Rab5 is not
necessary for the initial binding and tethering functions of EEA1. A
role for Rab5 binding was revealed, however, upon comparison of
endosomes in cells expressing full-length wild-type or mutated EEA1.
The mutant full-length EEA1 caused the accumulation of endosome
clusters and suppressed the enlargement of endosomes caused by a
persistently active form of Rab5 (Rab5Q79L). In contrast, expression of
wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome
tethering depends on the interaction of EEA1 with PtdIns-3-P, and its
interaction with Rab5 appears to regulate subsequent fusion.
Protein trafficking in the secretory and endocytic pathways
requires the coordinated participation of distinct protein classes, among which are tethering proteins and Rab GTPases. In the endocytic pathway the small GTPase Rab5 has been proposed to play key roles in
clathrin-coated vesicle formation, the regulation of endosome motility,
and early endosome fusion (1, 2). The specific mechanisms by which
these steps are regulated by Rab5 is not fully understood, but the
regulation of endosome fusion by Rab5 appears to be dependent on the
actions of the cytoplasmic protein
EEA1.1 EEA1 in turn has been
postulated to mediate the obligatory docking or tethering steps
required prior to homotypic endosome fusion, as well as to participate
in the fusion process itself (3, 4).
EEA1 is a large ~170-kDa polypeptide that contains extensive regions
of coiled-coil as well as a conserved Zn2+ finger motif at
its extreme COOH terminus, called the FYVE domain (5). The FYVE domain
interacts specifically with PtdIns-3-P, the most abundant product of
PI3-kinase activity in eukaryotic cells (6, 7), and is crucial for the
interaction of EEA1 with endosomal membranes (8). The dual interaction
of EEA1 with PtdIns-3-P and Rab5 has been hypothesized to confer
increased specificity and stability to the association of EEA1 with
endosomal membranes (3, 4). To directly test this hypothesis we have identified critical residues required for the interaction of the EEA1
COOH-terminal domain with Rab5, and documented the effect of
mutation of these residues on EEA1 localization and endosome fusion
dynamics. The results shown here suggest a temporal order of events in
which endosome tethering is mediated by the interaction of EEA1 with
PtdIns-3-P and subsequent endosome fusion is dependent on the
interaction of EEA1 with Rab5.
Plasmids and Recombinant Proteins--
Gst1277 was generated as
described previously (6). Site-directed mutations within this plasmid
were generated by polymerase chain reaction using Vent Polymerase (New
England Biolabs), and the resulting mutants were sequenced for
verification. 10xHis-Rab5c and Gst-Rab5c plasmids and purified
recombinant protein production have been described previously (9). Both
GFP-1277wt and GFP-1277dm were constructed by polymerase chain reaction
from either wild-type or double mutated Gst1277 and cloned in frame
into the SalI-BamHI sites in pEGFP-C1
(CLONTECH). RFP-Rab5Q79L was generated by
polymerase chain reaction from the full-length cDNA (a gift from D. Lambright) and cloned in frame into the
EcoRI-SalI sites in pDsRed1-C1
(CLONTECH). EEA1wt (full-length EEA1 cDNA in
pCMV5) has been described elsewhere (6). The cDNA encoding GFP was
subcloned into the EcoRI-XbaI sites 5' to
full-length EEA1 cDNA to generate GFP-EEA1wt. Both EEA1dm and
GFP-EEA1dm were constructed by insertion of nucleotides 3975-4233
PCR-amplified from GST1277dm into the PstI-BamHI
sites of EEA1wt or GFP-EEA1wt, respectively.
Cell Culture and Preparation of Cytosolic Extracts--
COS-7
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.). The
cells were grown in 100-mm dishes and transfected using LipofectAMINE
(Life Technologies, Inc.). To prepare cytosolic extracts, cells were
rinsed twice and swollen by incubation (10 min) in a 10-fold dilution
of cytosol buffer (25 mM HEPES, pH 7.0, 125 mM potassium acetate, 2.5 mM magnesium acetate,
0.2 M sucrose, 1 mM dithiothreitol, 1 mM ATP, 5 mM creatine phosphate, 0.01 mg/ml
creatine phosphokinase with proteinase and phosphatase inhibitors) in
water. Cells were homogenized by repeated passage through a
27-gauge needle and centrifuged at 1000 × g for
5 min to remove nuclei and unbroken cells. Supernatant was then
separated from particulate structures by centrifugation at 100,000 × g for 15 min for use in binding assays.
Binding Assays--
In vitro binding of 10xHis-Rab5c
to immobilized GST fusion proteins was performed as described
previously (9). Binding of cytosolic extracts from cells transfected
with either GFP-1277wt or GFP-1277dm was performed as follows. 5 µg
of GST-Rab5c fusion protein was bound to glutathione-Sepharose beads
that had been preblocked in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween
20). The beads were then incubated with 100 µM
GTP
Liposome binding assays were performed as described previously (6) with
minor modification. PS/PI liposomes were composed of 50%
phosphatidylserine and 50% phosphatidylinositol (Avanti), whereas PI3P
liposomes contained 1% PtdIns-3-P (Matreya), 50% phosphatidylserine,
and 49% phosphatidylinositol. 2.5 µg of cytosolic extract from COS-7
cells transfected with either GFP-1277wt or GFP-1277dm were treated
with 50 nM wortmannin for 5 min to inhibit endogenous
PI3-kinase. These extracts were then incubated with liposomes (100 µmol total lipid) for 15 min at room temperature in a 100 µl final
volume of 50 mM HEPES, pH 7.2, 100 mM NaCl, and
1 mM MgCl2. After centrifugation, liposome
pellets were analyzed by SDS-PAGE and immunoblotted with an anti-GFP
monoclonal antibody as described above.
Fluorescence Microscopy--
COS-7 cells were grown to 40-50%
confluence on coverslips and transfected using the calcium phosphate
precipitation method. 24 h post-transfection, cells were
either treated or untreated with 50 nM wortmannin
for 10 min as indicated or incubated with Texas red-labeled transferrin
(Molecular Probes) at a concentration of 25 µg/ml at 37 °C for the
indicated time. Coverslips were then washed twice with cold
phosphate-buffered saline (PBS), fixed in 4% formaldehyde/PBS for 10 min at 4 °C, and imaged using a conventional wide-field microscope
fitted with a 60× or 100× Nikon plan-apo objective. Images were
acquired with a cooled CCD camera (Hamamatsu) and MetaMorph software.
Coverslips labeled with antibody were fixed as described above followed
by permeabilization with 0.2% Triton X-100/PBS for 10 min at 4 °C.
Cells were then blocked with 1% fetal bovine serum in PBS for
30 min at 4 °C and stained in the same buffer with a monoclonal
antibody directed to the NH2 terminus of EEA1 (Transduction
Laboratories). Anti-mouse antibody coupled to fluorescein
isothiocyanate was used to detect the primary antibody.
To capture the information contained within the whole three-dimensional
volume of the cell, 18 optical sections at a resolution of 200 nm/pixel
and spaced at 250 nm intervals were taken. To remove out-of-focus blur
from each section, image restoration was performed using a iterative
constrained deconvolution algorithm described previously (10).
Morphometric analysis of endosomes was done on three-dimensional
images. Prior to analysis of each three-dimensional image, a threshold
intensity was set so that 33% of the light in the image would be above
threshold (this yielded a different threshold value for each
three-dimensional image). All voxels (voxel = pixel3) below the threshold intensity were set to 0 (background). The light above threshold was considered the signal in
the vesicles. A "vesicle" was defined as each group of
contiguous, above threshold voxels.
Live Cell Imaging--
For live cell imaging COS-7 cells were
transfected with EGFP-tagged constructs and after 24 h were imaged
using high speed, three-dimensional microscopy (10-13). The microscope
was configured to 133 nm/pixels using a 60× objective, and the laser
illumination was configured to provide a 488 nm excitation wavelength
with a flux on specimen of ~12 watts/cm2. Exposure times
of 5 ms were used to acquire each of 18 optical sections, spaced by 250 nm. Each set of 18 optical sections was acquired in less than 1 s,
allowing 20 ms for each 250-nm shift in focus. Stacks were acquired
every 10 s for 20 continuous minutes. The haze originating from
light sources outside the in-focus plane of the cell was reduced by
image restoration. Stacks were projected into single two-dimensional
images, which were concatenated into a QuickTime video format.
To dissect the specific structural requirements for the
interaction of EEA1 with Rab5, we built upon previous studies
demonstrating that, whereas the FYVE domain of EEA1 is necessary and
sufficient for its interaction with PtdIns-3-P, the FYVE domain and an
additional stretch of 30 amino acids NH2-terminal to the
FYVE domain are necessary for Rab5 binding (9). The structure of this
Rab5 binding region, a parallel coiled-coil homodimer depicted in Fig. 1A, was derived from the
crystal structure of the EEA1 COOH-terminal region bound to inositol
1,3-diphosphate (28). This region contains three glutamine residues
positioned on the outer surface of a helix, which may potentially
contribute to the interaction of the COOH terminus of EEA1 with Rab5.
To test this, we introduced point mutations converting these
glutamine residues, singly or in combination, to alanine and then
examined the ability of the resulting mutants to interact with Rab5
in vitro.
Mutation of glutamine 1328 to alanine substantially reduced, whereas
mutation of glutamine 1338 virtually abolished, the interaction between
the COOH terminus of EEA1 and Rab5. The combination of these two point
mutations completely ablates Rab5 binding. Interestingly, mutation of
glutamine 1335 significantly enhanced this interaction (Fig.
1B). None of the point mutations altered the function of the
FYVE domain as assessed by liposome binding assays (not shown). Thus,
these mutations result in a selective impairment of the interaction of
EEA1 with Rab5.
To examine how the inability of EEA1 to bind Rab5 would influence its
membrane localization, the subcellular localization of GFP-tagged
constructs encoding the wild-type COOH terminus of EEA1 (GFP-1277wt) or
the COOH terminus harboring glutamine to alanine mutations in amino
acids 1328 and 1338 (GFP-1277dm) was investigated. Both constructs
efficiently localized to endosomes (Fig.
2A, left panels). In both
cases, treatment of cells with the PI3-kinase inhibitors wortmannin
(Fig. 2A, right panels) or LY49653 (not illustrated)
abolished binding to endosomal membranes.
Sequential Roles for Phosphatidylinositol
3-Phosphate and Rab5 in Tethering and Fusion of Early Endosomes via
Their Interaction with EEA1*,
,, and Biochemistry and
Pharmacology, University of Massachusetts Medical School,
Worcester, Massachusetts 01615
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S or GDP
S in buffer A (20 mM HEPES, pH 7.2, 100 mM NaCl, 0.5 mM MgCl2, 2 mM EDTA, and 1 mM dithiothreitol) at 30 °C
for 30 min followed by the addition of MgCl2 to a final
concentration of 20 mM. Beads were then washed and
incubated with 2.5 µg of cytosolic extract for 1 h at 4 °C in
a final volume of 300 µl of cytosol buffer containing 10 mg/ml bovine
serum albumin and 0.1% Tween 20. Following centrifugation, the pellets
were washed four times in 1 ml of buffer A, 20 mM
MgCl2, 0.1% Tween 20. Bound proteins were resolved by
SDS-PAGE and transferred to nitrocellulose membrane, which was
blocked in 5% dry milk, TBST followed by incubation with
anti-GFP monoclonal antibody (Zymed Laboratories
Inc.). Filters were then incubated with horseradish
peroxidase-conjugated goat anti-mouse antibody (Promega), which was
detected by Renaissance enhanced luminol reagent (PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Point mutation of the Rab5 binding
region within the EEA1 COOH terminus. A, structure of
amino acids 1320-1340 of EEA1 based on the crystal structure of the
homodimeric EEA1 COOH-terminal region bound to Ins-1,3-P2,
as described by Dumas et al (28). B, GST
fusion proteins of EEA1 containing the sequence indicated by the amino
acid residues were immobilized on glutathione-Sepharose beads. Beads
were then incubated with 10XHis-Rab5 previously loaded with either
GTP S or GDPS. Bound Rab5 was detected by Western blotting with an
anti-His antibody.
View larger version (62K):
[in a new window]
Fig. 2.
Targeting of the wild-type and mutant EEA1
COOH termini to endosomes in intact cells.
A, EGFP fusion constructs of EEA1 containing amino acids
1277-1411 (GFP-1277wt) and the same construct harboring
mutations of glutamines 1328 and 1338 to alanine
(GFP-1277dm) were transfected into COS-7 cells and after
24 h were fixed before (left panels) or after
(right panels) treatment with 50 nM
wortmannin for 10 min. Cells were fixed in 4% formaldehyde and imaged
using conventional wide-field fluorescence microscopy using a Nikon
100× objective. B, cytosolic extracts from cells
transfected with either GFP-1277wt or GFP-1277dm were incubated with
GST-Rab5 bound to Sepharose beads and previously loaded with either
GTP S or GDPS (top) or with liposomes containing
phosphatidylinositol (PI) or PtdIns-3-P (PI3P)
(bottom); C, no liposome control. Bound GFP
protein was detected by Western blotting with an anti-GFP
antibody.
To confirm that the expressed proteins indeed differentially interact with Rab5, cytosolic extracts of cells expressing either GFP-1277wt or GFP-1277dm were incubated with immobilized GST-Rab5. To asses the function of the FYVE domain, similar extracts were incubated with liposomes containing PtdIns-3-P. Bound material was analyzed by immunoblotting with antibody to GFP. Although there was no difference in the ability of these constructs to associate with PtdIns-3-P liposomes in vitro, the mutant was deficient in Rab5 binding (Fig. 2B), indicating that the endosomal localization of the COOH terminus of EEA1 is mediated through its interaction with PtdIns-3-P and is independent of its interaction with Rab5. It is important to note, however, that the FYVE domain of EEA1 alone (amino acids 1336-1411) is not sufficient for endosome localization in vivo (9). This is most likely due to the inability of the FYVE domain to oligomerize rather than to specific loss of Rab5 binding. In support of this, sedimentation equilibrium experiments demonstrate that the FYVE domain alone (amino acids 1336-1411) is monomeric in solution, whereas the longer COOH-terminal construct, which contains a region of coiled-coil, forms dimers crucial for high affinity membrane binding (not illustrated).
It has been shown that overexpression of the EEA1 COOH terminus results
in the formation of unusually small early endosomes and blocks endosome
enlargement induced by the GTPase-deficient Rab5, Rab5Q79L (4).
Membrane localization is required for this effect, as point mutation of
the FYVE domain within this construct restores the Rab5Q79L-induced
endosome enlargement (14). It has been suggested that EEA1 functions
downstream of Rab5 in endosome fusion because of this effect. To
examine the requirement for the EEA1-Rab5 association in this assay,
RFP-tagged Rab5Q79L was co-transfected into COS-7 cells with either
GFP-1277wt or GFP-1277dm. Both GFP proteins co-localized with Rab5Q79L
on endosomal membranes (Fig. 3,
left) and when overexpressed blocked the enlargement induced
by Rab5Q79L (Fig. 3, right). This would indicate that the
inhibitory effect of the EEA1 COOH terminus on Rab5-induced endosome
enlargement occurs independently of its direct interaction with Rab5
and is most likely due to the inability of these deletion constructs to
allow endosome tethering to occur. Thus, although EEA1-mediated
tethering is crucial for endosome fusion, this function of EEA1 is
independent of its interaction with Rab5.
|
If both membrane localization and EEA1-mediated tethering are
independent of Rab5 association, the physiological role of the interaction between EEA1 and Rab5 remains unknown. To attempt to
elucidate this role, we examined the effects of overexpressing full-length wild-type (GFP-EEA1wt) or mutated (GFP-EEA1dm) EEA1 on
endosome function in COS-7 cells. To confirm that the expressed proteins indeed differentially interact with Rab5, cytosolic extracts of cells expressing either construct were incubated with immobilized GST-Rab5. This experiment was necessary, as the
NH2-terminal domain of EEA1 has been reported to be capable
of binding Rab5 in vitro (3). However, our experiments
indicate that the Q1328A and Q1338A mutation greatly impaired
the binding of full-length EEA1 to Rab5 (Fig.
4). The small residual binding observed
is likely to be because of the oligomerization of some of the mutant
EEA1 with the wild-type endogenous molecule (15). Thus, the
contribution of the NH2 terminus of EEA1 to Rab5 binding
appears to be minimal in these experiments.
|
GFP-EEA1wt was localized mainly in single vesicular structures
distributed throughout the cytoplasm, with an area of concentration toward the perinuclear region where larger, ring-like endosomes could
be seen (Fig. 5, left panels).
In contrast, GFP-EEA1dm was found in large clusters distributed
throughout the cytoplasm (Fig. 5, right panels). These
clusters represent tightly packed individual endosomes, clearly visible
in the enlarged inset region. Similar endosome clustering has been
observed in vitro under conditions in which SNARE priming is
impaired (3). Thus, the accumulation of nonfused endosome clusters in
cells overexpressing GFP-EEA1dm suggests a role for the interaction
between EEA1 and Rab5 in the regulation of endosome fusion following
tethering.
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We next examined the dynamic behavior of endosomes in living cells
expressing GFP fusion proteins of wild-type or mutant full-length EEA1.
GFP-EEA1wt was present on endosomes of varying diameter, many of which
had a characteristic ring-like appearance, probably because of their
relatively large, spherical morphology combined with the optical
properties of the wide-field microscope. These ring-like endosomes have
been observed by immunofluorescence of endogenous EEA1 in several
different cell types (16). The dynamic behavior of a group of such
endosomes in a cell expressing GFP-EEA1wt is illustrated in Fig.
6A. In this sequence, three
endosomes appear to come into close proximity with each other, form a
tight cluster, and fuse into a single, larger structure. Comparison of
the radius of the smaller and large final endosomes indicates an
increase consistent with the fusion of three smaller endosomes into
one. In cells expressing GFP-EEA1dm, these fusion events were not
observed. Instead, clusters of four or more large endosomes were
abundant, and these failed to fuse within the imaging time frame (Fig.
6B). These results suggest that the inability of Rab5 to
bind the mutant EEA1 protein results in a decrease in the efficiency of
homotypic endosome fusion.
|
To further test this hypothesis, we analyzed the behavior of endosomes
in cells co-expressing RFP-Rab5Q79L and either GFP-EEA1wt or
GFP-EEA1dm. As expected, expression of RFP-Rab5Q79L resulted in the
formation of enlarged endosomes, an effect that was significantly enhanced by co-expression of GFP-EEA1wt (Fig.
7, upper panels). In contrast,
coexpression of GFP-EEA1dm blocked the endosome enlargement induced by
RFP-Rab5Q79L (Fig. 7, lower panels). These results suggest
that the direct binding of Rab5 to the COOH terminus of EEA1 is
required for endosome fusion at a step subsequent to tethering.
|
To assess the functional consequences of the altered endosome fusion
dynamics induced by overexpression of EEA1 constructs, the trafficking
of Texas red-transferrin was analyzed (Fig.
8). When nontransfected cells or cells
expressing GFP-EEA1wt were incubated with Texas red-transferrin for 40 min, intense transferrin fluorescence was detected in a tight
pericentriolar region, which represents the recycling endosome. When
the intensity scale was set so as not to saturate the signal in the
pericentriolar region, the transferrin signal was virtually
undetectable in the GFP-containing endosomes. In contrast, after a
similar uptake period, the transferrin signal detected in the
pericentriolar region of cells expressing GFP-EEA1dm was comparable
with that detected in GFP-containing endosomes, indicating a delay in
traffic to the pericentriolar recycling endosome. In cells expressing
GFP-1277wt, in which endosome tethering is impaired, little transferrin
signal was detected in the recycling endosome after a 40-min uptake,
and extensive co-localization of Texas red-transferrin and GFP-1277wt
was observed. These results suggest that the transit of transferrin
through the early endosome to the recycling endosome is facilitated by EEA1-mediated endosome tethering and by fusion events that require the
direct interaction between EEA1 and Rab5.
|
To rule out that any of the observed phenotypes were due to the
insertion of a GFP-tag, cells expressing nontagged full-length EEA1wt
and EEA1dm were analyzed. Transfected cells could be identified by the
increase in fluorescence intensity after staining with anti-EEA1
antibody. Neither the number nor the size distribution of endosomal
structures in cells expressing EEA1wt at up to 15 times over the
endogenous level (Fig. 9) differed
appreciably from nontransfected cells. Cells overexpressing EEA1dm at
similar levels (5-15 times over endogenous level) differed from those overexpressing EEA1wt in that enlarged particles were observed throughout the cytoplasm (Fig. 9B). These particles
represent endosome clusters, as no single, large lumen was observed.
Thus, the introduction of the GFP tag on the EEA1 NH2
terminus appears not to alter its function measurably. Furthermore,
these results support the hypothesis that the accumulation of vesicle
clusters is due to the impairment of EEA1 binding to Rab5.
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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
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The process of endocytosis in eukaryotic cells plays a fundamental role in the control of cellular homeostasis. Through endocytosis, cells accumulate critical nutrients and regulate the surface complement of receptors. Thus, understanding the molecular basis of the endocytic process has been an important goal for cell biologists for many years. In vitro assays that measure the ability of endosomes containing exogenously added labels to fuse with each other have been used extensively to identify and characterize key elements involved in endosome fusion. Using these assays, the activities of PI3-kinase and of the GTPase Rab5 were found to be essential for endosome fusion in vitro (17-19). The discovery that EEA1 can interact with both PtdIns-3-P, a product of PI3-kinase, and activated Rab5 suggested that it may be an effector protein for these regulatory molecules. A model consistent with available results has been proposed in which PtdIns-3-P and Rab5 function cooperatively to recruit EEA1 to early endosomes (3, 4), where EEA1 then interacts with SNARE proteins to mediate endosome fusion ((20, 21). The goal of the present work was to test this model and to more precisely define the respective roles of the interactions between EEA1 and PtdIns-3-P and EEA1 and Rab5 in endosome function. The results shown here indicate that the interaction of EEA1 with PtdIns-3-P is necessary and sufficient for binding to endosomal membranes. Once bound, EEA1 may serve to link Rab5 activity to the machinery that regulates homotypic endosome fusion.
In vitro, EEA1 has been shown to interact with Rab5 through two regions, one immediately adjacent to the FYVE domain and an additional NH2-terminal binding site (3). However, mutation of the FYVE domain of EEA1 completely abolishes its interaction with endosomes (8), suggesting that the NH2-terminal binding domain is not likely to play a significant role in endosome binding in vivo. Furthermore, overexpression of the NH2 terminus of EEA1 lacking the FYVE domain does not compete for binding of endogenous EEA1 to endosomes (not shown). Results shown here indicate that although wild-type EEA1 bound immobilized Rab5, EEA1 constructs containing two point mutations adjacent to the FYVE domain did not (Fig. 4). Thus, the physiological significance of the in vitro interaction of the NH2 terminus of EEA1 with Rab5 remains to be determined.
Despite the failure of mutant EEA1 constructs to interact with Rab5, these constructs bound to endosomes to an extent indistinguishable from the wild-type constructs and in a wortmannin-sensitive manner. The most logical interpretation of these data is that the interaction of EEA1 with Rab5 is not required for the initial binding of EEA1 to early endosome membranes. What then is the role for the interaction between EEA1 and Rab5? Results shown here suggest that this interaction is required to regulate homotypic endosome fusion. Two lines of evidence support this hypothesis. First, endosomes in cells expressing the full-length mutant are found frequently in clusters, reminiscent of the clustered, unfused structures seen in yeast upon disruption of Vps9p (22, 23), a guanine-nucleotide exchange factor for Vps21p, which is a homologue of Rab5 (24). Second, the potent effect of persistently active mutants of Rab5 to accumulate large endosomes is impaired in cells expressing the full-length EEA1 mutant that cannot bind Rab5. These experiments suggest that Rab5 cooperates with EEA1 in the regulation of endosome fusion after EEA1 has bound. This model is consistent with genetic evidence in yeast for the molecular organization of an analogous pathway; Vac1p, the yeast orthologue of EEA1/Rabenosyn5, associates with the Rab5 homologue Vps21p and with Vps45p, a Sec1 homologue that binds and regulates the activity of the yeast SNARE Pep12p. In this system, the requirement for the GTPase is subsequent to membrane tethering.
The results presented here also raise a number of important issues. One in particular is that endosomes from cells expressing wild-type, mutant, or truncated EEA1, although morphologically distinct, all accumulate transferrin. Thus, the traffic of transferrin receptor from the plasma membrane to early endosomes that contain EEA1 does not appear to depend on the function of EEA1. The only consequence of disrupted EEA1 function on transferrin traffic appears to be a delay in the movement of transferrin from early endosomes into the perinuclear recycling compartment. The finding that transferrin traffic into endosomes occurs independently of EEA1 function is consistent with the inability of wortmannin, a potent PI3-kinase inhibitor, to inhibit transferrin uptake (25, 26). Thus, much more work is required to elucidate the mechanisms that control the vectorial delivery of plasma membrane-derived vesicles to endosomes.
In conclusion, the results presented here, derived from experiments in
intact cells, suggest a model for the spatial and temporal order of key
biochemical events in early endosome fusion. In this model, tethering
and fusion are regulated by the sequential interaction of EEA1 with
PtdIns-3-P, to mediate tethering, and with Rab5, to mediate fusion. The
requirement for tethering factors acting in conjunction with Rab
GTPases in other membrane transport steps (27) suggests that
interactions similar to those presented here may temporally and
spatially control multiple intracellular trafficking pathways.
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FOOTNOTES |
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* This work was funded in part by National Institutes of Health Grant DK54479 (to S. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains two QuickTime videos showing
endosome dynamics in live cells expressing either wild-type or mutant
GFP-EEA1.
¶ To whom correspondence should be addressed: 373 Plantation St., Worcester, MA 01615. Tel.: 508-856-6898; Fax: 508-856-1617; E-mail: silvia.corvera@umassmed.edu.
Published, JBC Papers in Press, October 15, 2001, DOI 10.1074/jbc.M109239200
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ABBREVIATIONS |
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The abbreviations used are:
EEA1, early endosome
antigen 1;
GFP, green fluorescence protein;
EGFP, enhanced green
fluorescence protein;
RFP, red fluorescent protein;
PtdIns-3-P, phosphatidylinositol 3-phosphate;
GST, glutathione
S-transferase;
wt, wild type;
dm, double mutated;
PI3-kinase, phosphatidylinositol 3-kinase;
PBS, phosphate-buffered
saline;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
GDPS, guanyl-5'-yl thiophosphate;
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
FYVE, Fab1p, YOTB, Vac1p, EEA1.
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ABSTRACT
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DISCUSSION
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