1 Graduate Program of Neuroscience, Weill Medical College of Cornell University,
1300 York Avenue, New York, NY 10021, USA
2 Department of Ophthalmology, Weill Medical College of Cornell University, 1300
York Avenue, New York, NY 10021, USA
3 Department of Biochemistry, Weill Medical College of Cornell University, 1300
York Avenue, New York, NY 10021, USA
4 Department of Cell and Development Biology, Weill Medical College of Cornell
University, 1300 York Avenue, New York, NY 10021, USA
* Author for correspondence (e-mail: chsung{at}mail.med.cornell.edu)
Accepted 18 September 2002
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Summary |
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Key words: Rab5, FYVE domain, SARA, Transferrin, EEA1, Endosome
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Introduction |
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One of the best-characterized Rab proteins, Rab5, is mainly localized on
early endosomes (Gorvel et al.,
1991). Rab5 is known to be involved in both clathrin-coated
vesicle-mediated transport from the plasma membrane to early endosomes and for
homotypic early endosome fusion (Bucci et
al., 1992
; Gorvel et al.,
1991
). Overexpression of a GTPase-deficient Rab5 mutant Rab5Q79L
resulted in enlarged endosomes, likely due to excess membrane fusion
(Barbieri et al., 1996
;
Bucci et al., 1992
;
Stenmark et al., 1994
).
Furthermore, Rab5Q79L overexpression interferes with both receptor-mediated
and fluid-phase endocytosis (Bucci et al.,
1992
; Li et al.,
1995
). So far, several Rab5 effectors have been isolated
(Zerial and McBride, 2001
).
Among them, p150 and p110ß are the regulatory subunit and
catalytic subunit, respectively, of 2 different phosphatidylinositol (PI)
3-kinases (Christoforidis et al.,
1999b
). Interestingly, 2 other Rab5:GTP effectors early
endosome autoantigen 1(EEA1) (Simonsen et
al., 1998
) and Rabenosyn-5
(Nielsen et al., 2000
) contain
a double zinc-finger FYVE (Fab1p, YOTB, Vac1p, and EEA1) domain, which is
known to bind to the phosphatidylinositol 3-phosphate (PtdIns(3)P),
lipid product of PI3 kinase, with high specificity
(Burd and Emr, 1998
;
Lawe et al., 2000
;
Stenmark and Aasland, 1999
).
It has thus been proposed that the Rab5:GTP as well as PtdIns(3)P generated on
early endosomes provide a dual binding mechanism for the recruitment of EEA1
and Rabenosyn-5. In in vitro endosome fusion assays, both depletion of either
EEA1 or Rabenosyn-5 inhibits this process
(Lawe et al., 2000
;
Nielsen et al., 2000
;
Simonsen et al., 1998
). In
addition to their role in endosome fusion, EEA1 and Rabenosyn-5 exhibit their
own unique structural and functional roles. For example, Rabenosyn-5, but not
EEA1, associates with the Sec1 homologue hVPS45 and participates in
endosome-lysosome trafficking (Nielsen et
al., 2000
).
SARA (Smad anchor for receptor activation) is also an FYVE domain protein.
SARA was initially reported as a Smad2 interacting protein with roles in the
recruitment of Smad2 and Smad3 to the ligand bound transforming growth factor
beta (TGF-ß) receptor. Thus, it plays a role in downstream signal
transduction (Tsukazaki et al.,
1998). Although overexpressed SARA has been shown to be located on
endosome-like vesicular structures in cultured cells
(Panopoulou et al., 2002
;
Seet and Hong, 2001
;
Tsukazaki et al., 1998
), the
exact localization of endogenous SARA and whether SARA plays role in membrane
trafficking have yet been fully investigated. In the present study, we
demonstrate that endogenous SARA is localized to the early endosome, however,
SARA labeling is not completely colocalized with EEA1 labeling. SARA
overexpression induced endosomal enlargement and inhibited the recycling of
transferrin (Tf) and reduced surface Tf receptor (TfR), phenotypes that have
been described for Rab5:GTP overexpression
(Stenmark et al., 1994
). Both
the morphological and functional changes caused by SARA could be corrected by
co-expression of Rab5:GDP. These results collectively suggest that SARA is a
novel player in the Rab5-regulated endosome trafficking pathway.
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Materials and Methods |
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The following antibodies were used in this study: FLAG mAb (cloneM2; Sigma), EEA1 mAb (Transduction Laboratories, Lexington, KY), Rab5A rabbit antibody (S-19; Santa Cruz Biotechnology Inc, Santa Cruz, CA), and antibodies against the extracellular (B3/25; Chemicon) and the cytoplasmic (H68.4; Zymed) domains of the human TfR. Alexa-488 and Alexa-594 conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Cy5 conjugated anti-mouse IgG was purchased from Jackson ImmunoResearch Lab (West Grove, PA).
Cloning and constructs
In a previous study, we isolated positive clones from a two-hybrid screen
of a bovine retinal cDNA library by using the C-terminal 39 residues of human
rhodopsin as bait (Tai et al.,
1999). One of these clones, when searched against the
GenBankTM database, encoded a partial sequence with a high degree of
conservation to human SARA (Tsukazaki et
al., 1998
). This bovine SARA cDNA fragment was then used as a
probe to screen a human retinal cDNA library in lambda gt10 (a kind gift of J.
Nathans, Johns Hopkins University, School of Medicine, Baltimore, MD).
Positive plaques were purified, and the cDNA inserts were cloned into
pBluescript IIKS (Stratagene, La Jolla, CA) for sequencing. Sequences derived
from a number of overlapping clones confirmed that they were human SARA cDNA
fragments (Tsukazaki et al.,
1998
). Full-length SARA was subsequently generated by fusing 4
cDNA fragments into the eukaryotic expression vector pRK5. FLAG-tagged SARA
and GFP-SARA were generated by placing full-length SARA 3' to the FLAG
peptide or EGFP, respectively. Detailed cloning procedures are available upon
request. Expression vectors of Rab5, Rab5Q79L, and Rab5S34N were kind gifts
from A. Francesconi (Albert Einstein Medical School, New York, NY).
Antibody production
A baculoviral expression vector encoding the human SARA fragment was
generated by inserting the cDNA fragment encoding D112-V1324 of SARA into the
pFastBacHTa vector (Invitrogen). Baculoviral protein encoding the His-tagged
SARA peptide was produced in Sf9 cells and purified using a nickel column
following the manufacturer's instructions.
Purified protein was then used as an immunogen for the production of rabbit
antiserum (Cocalico, Reamstown, PA). To remove cross-reactivities, we passed
the immunized serum through Sepharose columns conjugated with Sf9 cell lysates
and His-tagged GST fusion proteins. The final flow-through was then
affinity-purified on the His-tagged SARA conjugated Sepharose column, eluted
with 0.1 M glycine, pH 2.8, and neutralized with Tris-Cl, pH 9.5. The
resulting affinity-purified antibody is used for all experiments. Rabbit
anti-SARA antibody recognized a 140 kDa protein band from FLAG-SARA
transfected human embryonic kidney (HEK) cell lysates. A protein band with the
same molecular weight was also recognized by anti-FLAG antibody. SARA antibody
did not recognize EEA1, which was identified by anti-EEA1 antibody as a
180 kDa protein band on the same immunoblot.
Transfection and immunofluorescent staining of cell cultures
For transient transfection, HEK 293T cells and MDCK were transfected and
immunolabeled as described previously
(Chuang and Sung, 1998).
Briefly, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized
with 0.04% saponin before antibody incubation.
PFA fixed, non-permeabilized cells were used to labeled surface TfR with anti-TfR antibody (B3/25), followed by Alexa594-conjugated anti-mouse antibody. Since there is no detergent was used, only the epitopes exposed on the cell surface had access to the antibody. In some experiments, after the primary antibody incubation, cells were washed, post-fixed, permeabilized, and incubated with anti-Rab5 antibody followed by the corresponding secondary antibodies for detection. The samples were examined on an Axioskop 2 epifluorescent microscope (Zeiss, Oberkochen, Germany) equipped with the appropriate filter sets, and images were captured by a SPOT 2 cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI). Alternatively, samples were examined by a confocal microscope (Zeiss LSM 510). Images were processed using Metamorph software and exported to Adobe Photoshop for printing.
The relative surface to total TfR ratio was determined as described in
(Lampson et al., 2000).
Briefly, cells grown on coverslips were incubated with Cy3-Tf (3 µg/ml) in
serum-free medium (220 mM sodium bicarbonate, 20 mM HEPES pH 7.4) for 2 hours
at 37°C to saturate the Tf/TfR pathway. The cells were subsequently
transferred onto ice, washed three times with cold PBS-C/M and fixed with 4%
PFA. The surface TfR was detected by B3/25 mAb followed by Cy5-conjugated
anti-mouse antibody. Fluorescence microscopy was performed with a DMIRB
inverted microscope (Leica, Deerfield, IL), with a cooled CCD camera
(Princeton Instruments, Trenton, NJ). Images were acquired using a 63x
1.32 NA oil immersion objective. For quantification, total fluorescence of
each fluorophore was summed over all cells in a field using Metamorph software
(Universal Imaging, West Chester, PA), and nonspecific fluorescence (signals
obtained from cells labeled only the Cy5-conjugated secondary antibody) was
subtracted. Cy5/Cy3 ratios were measures of surface TfR normalized for the TfR
expression level.
To generate stable HEK lines, 293S cells were transfected with the FLAG-SARA and neomycin expression vectors. Cells surviving in medium containing G418 (500 µg/ml) were selected by cloning rings, and positive clones were identified by immunofluorescent staining and immunoblot assays. The stable lines were maintained in DMEM/F12 medium supplemented with 5% calf serum and G418 (250 µg/ml). Based on the immunostaining results, more than 90% of the cells expressed FLAG-SARA in the early-passage cells. The percentage of FLAG-SARA expressing cells decreased during subsequent passages, presumably the toll of SARA overexpression. For experiments in this paper, we used cells before passage 5, at which time at least 70% of the cells are FLAG-SARA immunoreactive. Based on immunoblotting, we estimated that the level of SARA expression in the stable clone we used for this study is about 2-3-fold relative to the endogenous SARA.
Tf recycling assays
The Tf recycling rate was measured biochemically as described previously
(Johnson et al., 2001).
Briefly, cells stably expressing FLAG-SARA or HEK cells were plated in
gelatin-coated 24-well tissue culture plates for 2 days before the experiment.
Cells were incubated with 3 µg/ml 125I-Tf in serum-free medium
for 2 hours at 37°C to achieve steady-state occupancy of the TfR. Cells
were washed with medium 2 (150 mM NaCl, 20 mM HEPES pH 7.4, 1 mM
CaCl2, 5 mM KCl, 1 mM MgCl2) three times, followed by a
2 minute wash in mild acid wash solution (200 mM NaCl, 50 mM
2-(N-morpholino)ethanesulfonic acid, pH 5.0) and a 1 minute
incubation with medium 2 to release surface-bound Tf. Cells were subsequently
incubated in efflux medium (3 µg/ml unlabeled Tf and 100 µM iron
chelator desferroxamine in serum-free medium) at 37°C for 0, 5, 10, 15,
20, or 30 minutes. At each time point, the efflux medium was collected and the
cells were solubilized for gamma counting. The radioactivity in the efflux
medium represents the 125I-Tf released from the cells during the
chase incubation, and the cell-associated radioactivity is the Tf remaining
inside cells. Cells incubated with a 200-fold excess of unlabeled Tf were used
to determine nonspecific binding of 125I-Tf. The radioactivity of
the efflux medium and solubilization solution was corrected by subtracting the
average of the radioactivity in the background wells. To calculate the
recycling rate constants, time points up to 30 minutes were used. The
recycling rate constant is the slope of the natural logarithm plot of the
percentage of Tf remaining associated with cells versus time.
Tf uptake assays
For the uptake experiments, HEK cells transfected with GFP-SARA (or
together with Rab5S34N) were loaded with Cy3-Tf (3 µg/ml) in serum-free
medium for 6, 15, or 120 minutes at 37°C. Cells were then placed on ice,
washed with acidic buffer and then medium 2. Cells were subsequently fixed
with 4% PFA for visualization. In the double transfection experiments, a 5:1
ratio of Rab5S34N- and GFP-SARA-encoding plasmids was used to ensure all
GFP-SARA-positive cells were also Rab5S34N transfected. The expression of
Rab5S34N in all GFP-SARA-positive cells was confirmed by the immunostaining of
Rab5 on a duplicate coverslip.
To determine the Tf internalization rate constant, HEK control cells and
HEK cells stably expressing FLAG-SARA were plated in gelatin-coated 6-well
clusters. Cells were loaded with 125I-Tf (3 µg/ml) in prewarmed
serum-free medium for 2, 4, 6, 8, 10 minutes at 37°C and then transferred
to ice for washing. Cells were rinsed twice in medium 2, followed by a 5
minute incubation in mild acid wash solution and a 5-minute incubation in
medium 2. Cells were then solubilized in 1% Triton X-100 containing 0.1 N NaOH
for gamma counting. In addition, counts derived from cells incubated with
125I-Tf and excess unlabeled Tf (600 µg/ml) were considered as
background and cells incubated with 125I-Tf at 4°C for 2 hours
were considered as total surface labeling. The internalized vs. surface
125I-Tf were plotted over time to derive the internalization rate
calculation (Wiley and Cunningham,
1982). Four duplicates were used for each time point in each
experiment and three independent experiments were carried out.
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Results |
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SARA-mediated endosome enlargement requires active Rab5
In contrast to endogenous SARA, we found that the ectopically expressed
SARA was distributed on much larger vesicular compartments, which were also
Rab5- and EEA1-positive (data not shown). Consistent to the previous reports
(Panopoulou et al., 2002;
Seet and Hong, 2001
;
Itoh et al., 2002
), this
observation suggested that overexpressed SARA resulted in early endosome
expansion.
The ability of SARA to induce endosome enlargement is similar to the
phenotype described for the overexpression of a GTP hydrolysis-deficient
mutant of Rab5 (Rab5Q79L) in cell cultures
(Stenmark et al., 1994;
Barbieri et al., 1996
). To
determine the functional relationship between SARA and Rab5, MDCK cells were
cotransfected with GFP-SARA together with Rab5Q79L (Rab5:GTP) or Rab5S34N
(Rab5:GDP) and fixed for immunolabeling. In these experiments, Rab5Q79L was
extensively colocalized with SARA on the membranes of enlarged endosomes
(Fig. 2A-C). The sizes of these
enlarged endosomes (2-9 µm) were comparable to those induced by
overexpression of Rab5Q79L alone (data not shown). Although there was a great
heterogeneity among the vesicle sizes in these transfected cells, we
consistently found that the large vesicles induced by Rab5Q79L and GFP-SARA
overexpression were significantly larger than those induced by overexpressing
GFP-SARA alone [1-5 µm, compare Fig. 2A
vs. 2D (arrowheads)]. In striking contrast, the large endosomes
caused by GFP-SARA overexpression disappeared in the Rab5S34N cotransfected
cells (Fig. 2D, arrow).
Instead, in the double transfected cells, GFP-SARA was mainly detected in the
cytosol and on small membrane vesicles. These results indicated that Rab5:GTP
is a prerequisite for SARA-mediate endosomal membrane enlargement.
|
However, unlike the two FYVE domain proteins EEA1 and Rabenosyn-5, which
interact with Rab5:GTP directly (Simonsen
et al., 1998; Nielsen et al.,
2000
), no specific interaction between SARA and Rab5Q79L could be
detected despite our repeated attempts with both two-hybrid and
co-immunoprecipitation assays (data not shown). These results together
suggested that the effect of Rab5 mutants on SARA distribution was most likely
through indirect interaction, such as via the PtdIns(3)P, rather than
the direct interaction between Rab5 and SARA.
Tf trafficking was affected by SARA overexpression
To access the functional consequences of SARA overexpression, we examined
the endocytosis and recycling of Tf in SARA overexpressing cells. To examine
Tf uptake, GFP-SARA transfected HEK cells were incubated with Cy3-Tf at
37°C for various time points before the cells were fixed for
visualization. Within 6 minutes, Cy3-Tf was found in the early endosomes
peripheral to the plasma membrane throughout the untransfected cells
(Fig. 3A). In contrast, very
little or no Cy3-Tf was detected in the SARA overexpressing cells at this time
point. However, Cy3-Tf could be detected in the SARA-positive enlarged
endosomes 15 minutes after Tf uptake. After 2 hours of loading, Tf reached a
steady-state occupancy of the endocytic pathway and was distributed throughout
the early endosomes and the recycling endosomes in the untransfected control
cells. On the other hand, Tf was largely concentrated in the SARA-positive
endosomal compartments in the GFP-SARA transfected cells. Double labeling of
TfR showed that the internalized TfR was also primarily associated with Tf in
the SARA-positive compartments (data not shown), suggesting that Tf and TfR
remained associated in the expanded early endosomes.
|
To determine if the internalization rate of Tf was affected by SARA overexpression, we compared the internalization rate constant of HEK cells stably expressing FLAG-SARA and untransfected HEK cells. In these assays, cells were loaded with 125I-Tf for 2-10 minutes at 37°C before harvesting. The ratio of radioactivity derived from the internalized Tf to the surface Tf was plotted as a function of time to determine the Ki, the endocytic rate constant. One example is shown in Fig. 3C: a SARA stable line and the parental HEK cells internalized Tf at approximately the same rate. The values for Ki were 0.12±0.02 minute-1 and 0.13±0.02 minute-1 for SARA stable line and HEK cells, respectively (4 duplicates for each experiments and 3 independent experiments).
We next examined the amount of surface TfR by immunolabeling with B3/25 mAb, which recognizing the extracellular domain of TfR. As shown in Fig. 4A,B, the punctuate surface labeling of TfR was significantly reduced in the GFP-SARA overexpressing cells compared to the untransfected cells. Quantitative measurement (see Experimental Procedures) showed that whereas the total TfR receptor levels (i.e. 2 hour Tf uptake at 37°C) were almost identical between the SARA-overexpressing cells and control cells, GFP-SARA overexpressing cells only displayed about 70% of surface TfR relative to that of control cells (Fig. 3D). The reduced surface expression of the TfR in SARA expressing cells likely accounts for the failure to detect Tf uptake into SARA expressing cells during a 6 minute pulse with fluorescent Tf (Fig. 3A).
|
The reduction in surface TfR is not due to increased degradation of the TfR since the half-life of TfR is about the same in control cells and SARA-expressing stable cells (data not shown). Thus, the decrease in cell surface receptor does not reflect receptor degradation.
The above results collectively indicated that over-expression of SARA slows the return of TfR to the cell surface. To test this possibility, GFP-SARA transfected HEK cells were incubated with Cy3-Tf for 2 hours at 37°C to reach a steady-state, and then chased in the presence of excess unlabeled Tf and iron chelator desferroxamine for various time periods. Cells fixed immediately after the 2 hour-Cy3-Tf loading showed that the majority of Tf had accumulated in the SARA-containing enlarged endosomes. In control cells about half of the internalized Cy3-Tf was recycled into the medium during a 10 minute chase incubation (Fig. 5A). Cy3-Tf recycling was significantly inhibited by SARA overexpression at all chase time-points examined. After a 30 minute chase, there was essentially no Cy3-Tf detectable in the untransfected cells, whereas an appreciable amount of Cy3-Tf remained in the SARA-positive endosomal compartments (Fig. 5A). These results are consistent with the model that Tf recycling was inhibited by the overexpression of SARA.
|
To quantify the Tf recycling rate of SARA expressing cells biochemically,
we assayed the Tf efflux in SARA-expressing stable lines and HEK cells. In
these experiments, cells were first incubated with 125I-Tf for 2
hours at 37°C. After a mild acid wash to remove surface-bound ligand,
cells were incubated in medium containing desferoxamine and unlabeled Tf for
various time periods. Release of 125I-Tf into the medium was
calculated as a percentage of total intracellular Tf at 0 minute. As shown in
a representative experiment (Fig.
5C), the SARA stable line released Tf at a significantly lower
rate. In data pooled from 3 experiments, we observed the recycling constant of
125I-labeled Tf was approximately 32% lower in the SARA-expressing
cells compared to the control cells (Fig.
5D). The degree of Tf recycling rate reduction was consistent with
the degree of reduction of surface TfR levels (30%;
Fig. 3D) in SARA overexpressed
cells.
Rab5S34N overexpression suppresses SARA-mediated effects of Tf
trafficking
The phenotypes caused by SARA overexpression (i.e. reduced surface TfR, and
slower Tf recycling) were similar to those described for Rab5Q79L
overexpression (Stenmark et al.,
1994). To test whether SARA is involved in the Rab5-mediated
Tf/TfR trafficking, we asked whether co-expression of the dominant-negative
Rab5 mutant (Rab5S34N) can attenuate the phenotypes resulting from SARA
overexpression.
In the Tf uptake experiments, the amount of Cy3-Tf internalized into cells expressing both GFP-SARA and Rab5S34N (indicated by the cytosolic distribution of GFP-SARA) in a 6 minute pulse was undistinguishable from that in neighboring untransfected cells (Fig. 3B). This is in contrast to SARA singly transfected cells, in which little internal Tf is detected in a 6 minute pulse (Fig. 3A).
In the surface TfR labeling experiments, cells overexpressing Rab5Q79L, but
not Rab5S34N, displayed significantly lower levels of surface labeling of TfR
compared to the neighboring cells (Fig.
4C,D and Fig.
4E,F). This is consistent with the biochemical experiment results
of previous report (Stenmark et al.,
1994). In contrast to the GFP-SARA singly transfected cells, the
level of surface TfR labeling for Rab5S34N/GFP-SARA-overexpressing cells was
very similar to that of untransfected control cells (compare
Fig. 4A,B with
Fig. 4G,H). Finally,
co-expression of Rab5S34N with GFP-SARA redirected Cy3-Tf recycling back to
the surface with kinetics similar to those of control cells. For example, at
the 30-minute chase time point, almost all Cy3-Tf had exited from
Rab5S34N/GFP-SARA double transfected cells
(Fig. 5B).
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Discussion |
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The enlarged endosome phenotype of SARA resembles the phenotype described for cells overexpressing Rab5Q79L. Furthermore, SARA-mediated endosomal swelling can be reversed by the overexpression of Rab5S34N. These results strongly argue that Rab5:GTP is required for recruitment of SARA onto early endosomal membranes and, in turn, for the morphological and functional alteration of the early endosome. It is intriguing that the extent of endosome enlargement appears to be greater in the Rab5Q79L expressing cells than in the SARA expressing cells. The higher degree of endosome fusion caused by Rab5Q79L overexpression can be explained by the existence of multiple Rab5 effectors, which might cooperatively enhance the endosome fusion events. However, at the present time, there is no evidence to rule out the possibility that an alternative mechanism is involved for SARA-mediated endosome swelling. For example, while Rab5Q79L overexpression is proposed to enhance homotypic fusion between early endosomal membranes, excess SARA may disassemble the coated protein on early endosomes and thus inhibit budding.
Alteration in receptor-mediated endocytic trafficking by SARA
overexpression
In addition to morphologic changes in early endosomes, overexpression of
Rab5Q79L also causes a decrease in Tf recycling and the number of surface TfRs
(Stenmark et al., 1994).
However, the effector of these Rab5 functions has yet been identified. In the
present report, we showed that SARA overexpression affects Tf and TfR
trafficking in a similar fashion as Rab5Q79L overexpression. Furthermore, all
these phenotypes caused by SARA can be reversed by co-expression of Rab5S34N.
Taken together, these results argue for a functional link between SARA and the
Rab5-mediated endosomal trafficking pathway. Namely, the Rab5Q79L-mediated
Tf/TfR recycling delay is likely to be mediated through SARA. Nevertheless, we
do not refer to SARA as a Rab5 effector because all other Rab5 effectors
reported so far, but not SARA, can directly interact with Rab5 in in vitro
binding assays.
It has been shown that Rab5Q79L overexpression increases the Tf internalization rate, whereas SARA overexpression has little or no effect on the Tf internalization rate. This is consistent with the involvement of Rab5 in the transport of clathrin-coated vesicles from the plasma membrane, whereas SARA is not detectable on the plasma membrane.
The early endosome is a highly dynamic structure. Constant fusion and fission are required to maintain the homeostasis of early endosomal compartments. Abnormal accumulation of Tf and TfR in the SARA-induced large early endosomes, but not the recycling endosomes, suggests that perturbation of early endosome morphology may affect the temporal and/or spatial cues for the transit from early endosome to recycling endosome. This interference may explain the slowdown of Tf and TfR recycling back to the cell surface.
Endosomal localization of SARA
Several lines of evidence suggest that the endosomal membrane distribution
of SARA relies largely on the interaction between the FYVE domain and
PtdIns(3)P. First, SARA displayed a cytosolic distribution in
response to wortmannin (Panopoulou et al.,
2002). Second, deletion of FYVE domain
(Tsukazaki et al., 1998
;
Panopoulou et al., 2002
;
Itoh et al., 2002
) results in
a complete removal of its membrane localization. Finally, it has been reported
that the first conserved cysteine at position 603 is critical for the FYVE
domain structure (Gaullier et al.,
2000
; Seet and Hong,
2001
). GFP-SARA mutant GFP-SARAC603S, in which the
cysteine-603 residue in the FYVE domain was mutated to serine, showed a
completely cytosolic distribution (Y.H. and C.H.S., unpublished).
Recent data suggested that the FYVE domain of SARA (aa574-aa660) itself
binds to PtdIns(3)P with a high affinity (Kd=30
nM) (Panopoulou et al., 2002).
This affinity is higher than that detected between PtdIns(3)P and the
FYVE domain of EEA1 (50 nM) (Gaullier et
al., 2000
). This affinity difference may be sufficient to explain
why the FYVE domain of EEA1 (aa1336-aa1411) is not sufficient for its
endosomal localization (Lawe et al.,
2000
). Instead, the dual interaction of EEA1 with
PtdIns(3)P as well as Rab5 may be needed to increase specificity and
stability for the association between EEA1 and endosomal membranes
(Christoforidis et al., 1999a
;
Simonsen et al., 1998
;
Lawe et al., 2000
).
In contrast to the Rab5 effectors isolated so far, SARA did not display a detectable interaction to Rab5:GTP in yeast two-hybrid and co-immunoprecipitation assays (data not shown). Whether these negative results are resulted from weak or transient interaction between these two molecules or through a common binding partner is presently unclear. However, because our data showed that overexpression of Rab5:GDP suppresses the endosomal membrane localization of SARA as well as all the SARA-mediated functional phenotypes, we thus propose that Rab5:GTP stimulates the local enrichment of PtdIns(3)P on early endosomal membranes, and SARA is subsequently recruited to the early endosome based a single-mode interaction through PtdIns(3)P.
Within the past few years, increasing numbers of FYVE domain proteins have
been localized to early endosomes. Why are multiple FYVE domain proteins
needed by a single early endosome? And how do these FYVE domain proteins
participate in Rab5-mediated endosomal functions in a coordinated manner? In
the case of EEA1 and Rabenosyn-5, although both of them share roles in
endosome fusion, EEA1 is unable to rescue the endosome fusion suppression
caused by the depletion of Rabenosyn-5
(Nielsen et al., 2000). This
suggests that EEA1 and Rabenosyn-5 serve somewhat distinct roles in these
processes. In addition, Rabenosyn-5 plays a role in lysosomal trafficking of
cathepsin D, which EEA1 does not (Nielsen
et al., 2000
). One model that has been proposed is that there are
functional subcompartments on early endosomes, and each subcompartment
participates in a different aspect of endocytic trafficking
(De Renzis et al., 2002
). We
have observed that endogenous SARA and EEA1 are indeed distributed on distinct
microdomains of early endosomes. In addition, the stoichiometry of these two
molecules, indicated by the labeling intensity, also appears to vary among
different pools of early endosomes. These observations suggest the possibility
that different FYVE domain proteins may function differently based on their
spatially distinct distributions and the level of protein expression.
SARA, a functional link between signal transduction and vesicular
trafficking
SARA has been suggested to be involved in the TGF-ß receptor mediated
signal transduction pathway. SARA recruits Smad2 and Smad3 to the TGF-ß
receptor upon receptor stimulation. The Smads are then phosphorylated by the
activated receptor kinases. The phosphorylation of Smad2 and Smad3 enable them
to bind Smad4. The resulting heteromeric complex is subsequently translocated
to the nucleus, where it controls the transcription of target genes
(Tsukazaki et al., 1998).
Although ectopicically expressed SARA has been found on endosomes in previous
reports (Seet and Hong, 2001
;
Panopoulou et al., 2002
), the
present report, for the first time, confirms that the endogenous SARA is
indeed localized on early endosomes. Together with others
(Panopoulou et al., 2002
;
Itoh et al., 2002
), these
results support the model that the TGF-ß receptor signaling is taken
place on the endosomal membranes.
In addition to its signal transduction role, this report suggests that SARA
also plays an important role in the dynamic morphology and function of
endosomes, arguing that SARA is a molecule linking between membrane
trafficking and signal transduction on endosomes. Similar to SARA, the FYVE
domain protein-hepatic growth factor-regulated tyrosine kinase substrate (Hrs)
has also been linking between the TGF-ß/Smad signalling pathway and
endosomal membrane trafficking (Raiborg et
al., 2001b). Nevertheless, the endosomal targeting of Hrs is
via a Rab5-independepent pathway
(Raiborg et al., 2001a
).
In addition to its role in downregulating surface receptors, endocytosis
has been recently recognized as a mechanism tightly associated with the
signaling pathway. One prominent example is that the internalization of
G-protein coupled receptor (GPCR) is targeted to endosome, via the
ß-arrestin-and the clarthin-dependent pathway, on which ß-arrestin
recruites and activates certain members of mitogen-acitvated protein kinase
(MAPK) cascades (Pierce and Lefkowitz,
2001). Furthermore, it has been shown that Rab5 plays roles in the
internalization, endosomal sorting and recycling of a number of GPCRs
(Iwata et al., 1999
;
Seachrist et al., 2000
;
Seachrist et al., 2002
). It
would be of great interest to investigate whether SARA also participates roles
in the signal transduction pathways of GPCRs on endosomal compartments.
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Acknowledgments |
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References |
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