From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Received for publication, November 15, 2000, and in revised form, December 11, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current models for sorting in the endosomal
compartment suggest that endosomal geometry plays a significant role as
membrane-bound proteins accumulate in tubular regions for recycling,
and lumenal markers accumulate in large vacuolar portions for delivery
to lysosomes. Rab5, a small molecular weight GTPase, functions in the
formation and maintenance of the early/sorting endosome. Overexpression of the constitutively active form, Rab5(Q79L), leads to enhanced endosome fusion resulting in the enlargement of early endosomes. Using
an adenoviral expression system to regulate the time and level of
Rab5(Q79L) overexpression in HeLa cells, we find that although
endosomes are dramatically enlarged, the rates of transferrin receptor-mediated endocytosis and recycling are unaffected. Moreover, despite the enlarged endosome phenotype, neither the rate of
internalization of a fluid phase marker nor the rate of recycling of a
bulk lipid marker were affected. These results suggest that GTP
hydrolysis by Rab5 is rate-limiting for endosome fusion but not for
endocytic trafficking and that early endosome geometry may be a less
critical determinant of sorting efficiencies than previously thought.
Endocytic vesicles deliver their content of membrane proteins,
lipids, and lumenal content to the early or sorting endosomal compartment consisting of tubular and vacuolar portions. Many receptor-ligand complexes dissociate in the mildly acidic environment of the early endosome (1). It has been proposed (1, 2) that endosomal
morphology and resulting geometric considerations play a major role in
controlling sorting efficiency in the early endosome. In this model,
membrane proteins destined for recycling accumulate in long tubular
extensions of the early endosome, which have a high surface to volume
ratio. Fluid phase content including released ligands is deposited in
the vacuolar portions of the early endosome, which, being spherical,
approach a minimum surface to volume ratio. These vacuolar portions
dissociate from tubular regions to carry their contents to late
endosomes and/or lysosomes (3).
Rab5 is a small molecular weight GTPase associated with the plasma
membrane and early/sorting endosomes. Rab5 controls homotypic early
endosome fusion and thus functions in the formation of early endosomes
(4-6). A point mutation in the GTPase domain (glutamine to
leucine; denoted as Rab5(Q79L)) reduces Rab5 GTPase activity and
results in a mutant Rab5 with an increased propensity to be in the
active, GTP-bound state (7, 8). Expression of this constitutively
active form of Rab5 enhances homotypic endosome fusion leading to the
formation of enlarged early endosomes. It also has been reported that
Rab5(Q79L) overexpression increases the rate of transferrin receptor
uptake and decreases the rate of transferrin receptor recycling (8)
although the mechanism for these effects remains obscure.
Rab5 is preferentially associated with the vacuolar portions of the
early endosome (9), and Rab5(Q79L) overexpression leads to the
formation of large spherical endosomes as visualized in semithick
sections by electron microscopy (10). To test whether the Rab5(Q79L)
effects on transferrin receptor endocytosis and recycling can be
correlated with these dramatic changes in endosomal size and geometry,
we utilized a tetracycline-regulatable adenoviral expression system
that allows us to temporally control Rab5(Q79L) expression levels. The
early endosomal compartment was dramatically enlarged in adenovirally
infected HeLa cells overexpressing Rab5(Q79L). However, the presence of
these enlarged endosomes did not alter the kinetics of endocytic
membrane trafficking of either cell surface receptors or bulk membrane
lipids. These unexpected results argue that geometric considerations
may contribute to a lesser extent than previously assumed in
determining the sorting and recycling efficiencies of the early
endosomal compartment.
Cell Culture--
tTA-HeLa cells were cultured in
DMEM1 supplemented with 5%
(v/v) fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin (growth medium). Wild type and mutant (canine)
Rab5a constructs (a gift of M. Zerial) were tagged with the
hemagglutinin epitope on the amino terminus and subcloned into pUHD
expression vectors (11). HA-Rab5(WT)- and HA-Rab5(Q79L)-expressing
cells were generated by cotransfecting the tTA-HeLa cells with cDNA that encodes HA-Rab5(WT) or HA-Rab5(Q79L) (10 µg) and the plasmid pBSpac (0.5 µg) by calcium phosphate transfection. Positive clones were selected by culture in 200 ng/ml puromycin, 400 µg/ml G418, and
2 µg/ml tetracycline and screened by Western blot for their abilities
to express Rab5 48 h after induction by the removal of
tetracycline (11).
Stably transfected cells were cultured in the presence of 1 µg/ml
tetracycline. Wild type or mutant Rab5 expression was induced by
washing out the existing tetracycline with two PBS (phosphate-buffered saline, pH 7.4) washes and incubating the cells in growth medium without tetracycline for 48 h.
Alexa-Transferrin Labeling and
Immunofluorescence--
Cells, grown on coverslips to ~70%
confluency, were washed twice with room temperature PBS and then
incubated with 50 µg/ml Alexa-transferrin (Molecular Probes) in
PBS+++ (PBS, 1 mM MgCl2, 1 mM CaCl2, and 0.2% bovine serum albumin) for
the indicated times at 37 °C. Coverslips were moved to 4 °C, washed twice with ice-cold PBS++++ and three times with
ice-cold citrate buffer, and re-equilibrated with two additional
ice-cold PBS++++ washes (12). The coverslips were fixed in
a 4% formaldehyde/PBS++ (PBS, 1 mM
MgCl2, and 1 mM CaCl2) solution at
room temperature for 5 min and on ice for an additional 15 min. Excess
formaldehyde was removed with 3 × 5-min washes in
PBS++. Cells were permeabilized in 0.1% saponin/5% goat
serum/PBS++ for 15 min. After 3 × 5-min
PBS++ washes, the coverslips were incubated with primary
antibody for 1 h. Antibodies used (source in parentheses) were
mouse monoclonal anti-HA tag 12CA5 (Ian Wilson, The Scripps Research
Institute) and mouse monoclonal anti-Rab5 (Transduction Laboratories).
Unbound primary antibody was removed with 3 × 5-min
PBS++ washes, and the coverslips were incubated with the
appropriate secondary antibody (noted in figure legends). Coverslips
were subjected to 6 × 5-min PBS++ washes, rinsed in
Millipore water, and mounted on a coverslip slide using Fluoromount G
(EM Sciences).
Western Blot Detection of HA-Rab5--
35-mm dishes of cells
were washed twice in room temperature PBS and put on ice with PBS to
cool to 4 °C for 5 min. Cells were then harvested in 500 µl of
ice-cold solubilization buffer (150 mM NaCl, 1% Nonidet
P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and 2 mM
phenylmethylsulfonyl fluoride). Lysates were solubilized by gently
rocking for 10 min at 4 °C and centrifuged at 14,000 rpm in an
Eppendorf microcentrifuge for 10 min at 4 °C to pellet the insoluble
material. Protein concentration was determined by BCA, and 100 µg of
solubilized protein was run on 13% SDS-polyacrylamide gel
electrophoresis mini-gel, transferred to nitrocellulose, and
immunoblotted using monoclonal antibodies against Rab5 or 12CA5 as
above. Proteins were detected using secondary goat anti-mouse
antibodies conjugated to horseradish peroxidase and visualized using
enhanced chemiluminescence.
Adenovirus Generation--
HA-Rab5(WT) or HA-Rab5(Q79L) was put
under the control of a tetracycline-regulatable promoter in the pAdlox
vector 3' to the Adenoviral Infection--
In experiments in which cells
expressed Rab5 continuously for 18 h, tTA-HeLa cells at ~70%
confluency were infected with adenovirus at an m.o.i. of 10 plaque-forming units/cell. Cells were infected with adenovirus in DMEM
with or without 1 µg/ml tetracycline for 2 h at 37 °C. After
infection, the viral medium was removed and replaced with growth
medium with or without tetracycline.
When Rab5 expression was studied using a bolus concentration, tTA-HeLa
cells at ~70% confluency were infected with adenovirus at an m.o.i.
of 300 plaque-forming units/cell. Infection took place at 37 °C for
2 h in DMEM, and the adenovirus-containing medium was
removed and replaced with growth medium containing 10 µg/ml
cycloheximide for 3 h to prevent protein but not mRNA synthesis. Cycloheximide was washed out with three washes of DMEM, and
the cells were returned to 37 °C incubation with growth
medium for the indicated times, typically 1, 3, or 12 h. At
all time points, the cells remained adherent to the dish without any
obvious signs of toxicity or cell death.
Transferrin Uptake and Recycling--
Infected cells were
assayed for transferrin uptake and recycling as previously described
(14) with minor modifications. To measure a single round of transferrin
receptor endocytosis, harvested cells were preincubated for 1 h
with 4 µg/ml B-XX-Tfn. Prior to internalization, unbound ligand was
removed by three washes in ice-cold PBS++++ (PBS, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, and 0.2% bovine serum albumin, pH 7.4). Cells
were resuspended to a concentration of 3-4 × 106
cells/ml and incubated at 37 °C for the indicated times. B-XX-Tfn trafficking was stopped by transferring a 50-µl aliquot of the cell
suspension to an Eppendorf tube containing 750 µl of ice-cold PBS++++. Internalized B-XX-Tfn was determined using an
enzyme-linked immunosorbent assay-based assay as previously described
(14).
Horseradish Peroxidase (HRP) Uptake--
Fluid phase uptake was
performed on infected 35-mm dishes of cells as previously described
(15, 16).
Lipid Recycling--
Lipid recycling was performed as described
by Hao and Maxfield (17). Quantitation of C6-NBD-SM in
medium or in cell lysates was performed using a PerkinElmer Life
Sciences fluorimeter at an excitation wavelength of 465 nm and
measuring the peak height between 518 and 558 nm. Data are plotted as
the C6-NBD-SM that remains cell-associated at each time point.
Expression of HA-Rab5(Q79L) Induces Enlarged Endosomes in Stably
Transformed Cells--
To begin to probe the mechanism of Rab5(Q79L)
effects on TfnR endocytosis and recycling, we generated stably
transformed cell lines expressing HA-tagged wild type Rab5
(HA-Rab5(WT)) and constitutively active HA-Rab5(Q79L) under the control
of a tetracycline-responsive expression system. Using this system,
stable cell lines can be generated while avoiding any deleterious
effects that may result from continuously altering cellular membrane
trafficking (11). Stable cell lines generated in this manner express
either HA-Rab5(WT) or HA-Rab5(Q79L) in a tetracycline-regulated manner
as determined by Western blotting using either the 12CA5 anti-HA
antibody (not shown) or antibodies against Rab5, which reveal both the
more slowly migrating recombinant HA-tagged protein and the endogenous Rab5 (Fig. 1A). In these
stably transformed cells, recombinant HA-Rab5 is expressed at roughly
equimolar levels compared with endogenous protein. Expression of
Rab5(WT) at these levels did not affect endosome morphology
(Fig. 1B, upper left), whereas expression of
Rab5(Q79L) at these levels was sufficient to cause the expected
morphological phenotype Adenovirus-mediated, Tetracycline-regulated Expression of
Rab5(Q79L)--
Given that a threshold level of Rab5(Q79L) expression
may be required to cause changes in membrane trafficking (8), it remained possible that the lack of changes in TfnR trafficking was due
to low levels of exogenous protein expression. Consequently, we elected
to employ an adenoviral expression system to obtain reproducibly and
uniformly higher levels of overexpression. In addition, adenovirus
allows for the rapid induction of high levels of protein, thus
circumventing problems that may occur as a result of chronic exposure
of a foreign protein to the cell. The adenoviral expression system was
designed to retain the tetracycline regulation so that any potential
adenovirus effects could be controlled by infection of cells in the
presence of tetracycline.
When cultured under inducing conditions for 18 h in the absence of
tetracycline, tTA-HeLa cells infected with recombinant adenoviruses
encoding either WT or mutant Rab5 expressed 50-100-fold higher levels
of the desired protein compared with endogenous Rab5 (Fig.
2A). Importantly, WT and
mutant Rab5 expression was not detectable when infected cells were
cultured in the presence of tetracycline. As expected at these high
levels of overexpression, the characteristically enlarged endosomal
morphology was readily apparent in cells expressing HA-Rab5(Q79L) (Fig.
2B). These enlarged endosomes remained accessible to
internalized Tfn as indicated by the colocalization of fluorescently
labeled transferrin (Alexa-Tfn) and Rab5 containing vesicles stained
with an antibody that recognizes the HA epitope (Fig. 2B).
Despite the fact that transferrin receptors were trafficking through
these dramatically enlarged endosomes when the kinetics of transferrin
receptor trafficking was measured, there were no changes in the uptake
of the transferrin receptor (Fig. 2C) compared with control
uninfected cells or cells infected with HA-Rab5(WT). Like the stably
transfected Rab5 tTA-HeLa cell lines, adenovirus expression of wild
type and mutant Rab5 did not alter the steady state accumulation of Tfn
within the cell.
Transferrin Endocytosis and Recycling Are Unaffected by
HA-Rab5(Q79L) Overexpression--
Our findings are inconsistent with
previous results showing effects of both Rab5(WT) and Rab5(Q79L)
overexpression on endocytosis and recycling of TfnR (5, 8). One trivial
explanation for these differences is that previous studies were
performed on adherent cells following internalization of
125I-Tfn, whereas our assay follows B-XX-Tfn uptake
in nonadherent cells. However, similar results were obtained when we
assayed endocytosis and recycling of 125I-Tfn in adherent
adenovirally infected HeLa cells using the methodology of others (Refs.
5, 8, and data not shown). A second methodological difference was that
previous studies employed a protocol that ensured a rapid bolus of Rab5
overexpression (5, 8). In contrast, the persistent overexpression of
Rab5(Q79L) in our system may enable induction of a compensatory
mechanism(s) that restores transferrin receptor trafficking to normal
steady state rates. Therefore, we adapted our expression system for
rapid induction of a bolus of protein expression. For these
experiments, cells were infected with a 30-fold higher m.o.i. of
adenovirus (see "Materials and Methods" for details), and Rab5
expression was controlled using cycloheximide. Briefly, tTA-HeLa cells
were infected with adenovirus (m.o.i. of 300) for 2 h. Cells were
then treated with 10 µg/ml cycloheximide for 3 h to accumulate
mRNA in the absence of protein expression. The cycloheximide was
washed from the cells, and protein was expressed for the indicated
periods of time before experiments were performed. As shown in Fig.
3A, this protocol allows for
tight control of protein synthesis while permitting a regulatable, high
level of Rab5 expression. Within 1 h of HA-Rab5(Q79L) expression,
exogenous HA-Rab5(Q79L) levels were estimated to be 50-fold over
endogenous Rab5 (Fig. 3A). The level of HA-Rab5(Q79L)
expression continued to increase with increased time of incubation in
the absence of cycloheximide, plateauing at 6-12 h postinfection. A
similar expression pattern was seen when cells were infected with
HA-Rab5(WT) adenovirus (data not shown). After only an hour of protein
synthesis, the enlarged endosomal phenotype could be detected (Fig.
3B). Increased duration of HA-Rab5(Q79L) synthesis caused a
successive increase in the size of early endosomes, whereas increased
HA-Rab5(WT) expression caused only minor increases in endosome
size.
At each time point, we performed a kinetic analysis of transferrin
uptake and recycling (Fig. 4). Consistent
with our findings thus far, despite the dramatic changes in endosomal
morphology seen at even the earliest time points of HA-Rab5(Q79L)
expression (1 h), we were unable to detect changes in endocytosis or
steady state accumulation of TfnR within the cell. Although endosome size continued to enlarge at 3 and 12 h of HA-Rab5(Q79L)
expression, there was similarly no effect on the rates or efficiency of
TfnR uptake and intracellular accumulation compared with uninfected cells or cells infected in the presence of 1 µg/ml tetracycline, which served as controls. In all cases, Tfn uptake was maximal at 5 min, and recycling occurred with a half-time of ~7-8 min, consistent
with results of others (18-20).
Rates of Bulk Phase Endocytosis and Lipid Recycling Are Unaffected
by Trafficking through Enlarged Endosomes--
It has been proposed
(1, 2) that sorting in the early endosome occurs, at least in part, by
a default mechanism based on the geometry of the tubulovesicular early
endosome. In this model, the high surface area of the tubular portions
of the endosome facilitates recycling of membrane-associated
components, perhaps through an iterative process (19, 21). Others have
argued that more directed sorting mechanisms are required for the
highly efficient endocytic trafficking of recycling receptors such as the TfnR (22). The appearance of coated buds containing TfnR on early
endosomes (22) and the sorting motif-dependent inhibition of TfnR recycling by bafilomycin (23) support this latter hypothesis. Thus, our inability to detect an effect on the kinetics and efficiency of TfnR uptake and recycling in cells despite dramatic alterations in
early endosome size and geometry may reflect the involvement of
Rab5-independent, directed sorting events. Consistent with this
possibility, one notable difference in our experiments compared with
others is that in previous studies human TfnR vectors were introduced
in parallel with the Rab5(WT) and Rab5(Q79L) constructs (8), whereas we
are studying transport kinetics of endogenous receptors. Thus, it is
possible that at higher levels of expression TfnR endocytic trafficking
becomes more sensitive to alterations in endosomal morphology and/or
Rab5 function than that of endogenous Tfn receptors.
Importantly, we obtained similar results when examining endogenous TfnR
endocytosis and recycling kinetics using adenovirally infected HepG2
cells expressing Rab5(Q79L) (data not shown).
To determine whether the dramatic changes in early endosome morphology
affect the bulk sorting properties of early endosomes as predicted by
current models, we examined the kinetics of endocytosis of a bulk fluid
phase marker and the kinetics of recycling of a bulk membrane lipid
marker. To focus on the rates of volume endocytosis rather than the
extent of volume accumulation, we analyzed the initial rate of fluid
phase HRP uptake. As can be seen (Fig. 5,
A-C), we were unable to detect differences in the rate of
HRP endocytosis at either 1, 3, or 12 h after bolus induction of
expression of WT or mutant Rab5 compared with either uninduced or
uninfected controls. Previous studies on HRP uptake in Rab5-expressing cells (7) focused on later time points of uptake when changes in
endosomal volume will be reflected by increased accumulation of HRP at
steady state. Our results suggest that GTP hydrolysis by Rab5 is not
rate-limiting for bulk or receptor-mediated endocytosis in HeLa
cells.
We next measured the rates of bulk membrane recycling in cells
overexpressing Rab5(Q79L), expecting that membrane lipids would accumulate in the enlarged vacuolar portions of the early endosome slowing their recycling. For these experiments we used
C6-NBD-SM, a readily extractable, fluorescently labeled
membrane lipid (17). Briefly, cells were labeled with
C6-NBD-SM for 10 min at 37 °C to allow the
C6-NBD-SM to traffic to the early endosomes. After extracting the plasma membrane C6-NBD-SM through a series
of backwashes in a fatty acid-free bovine serum albumin solution,
dissociable C6-NBD-SM was measured by the fluorescence in
the medium. Unexpectedly, there was no appreciable difference in
the rate or extent of C6-NBD-SM recycling from the endosome
to the plasma membrane at any time point after induction of Rab5 WT or
mutant overexpression (Fig. 5,
D-F). Thus, in these cells efficiency of recycling
of either bulk membrane or TfnR was not affected by dramatic changes in endosome geometry.
Conclusions--
We find that the early endosomal compartment
significantly expands in cells overexpressing the constitutively active
Rab5 mutant, Rab5(Q79L). This finding is consistent with previous work of others (8, 24) and with the model that Rab5 plays a critical role in
early endosome biogenesis and morphogenesis by controlling the rate of
endosome fusion events while in the Rab5/GTP-bound form (4, 5).
Unexpectedly, and in contrast to previous reports (7, 8), in the
presence of Rab5(Q79L) overexpression we observed no detectable
acceleration in the kinetics of Tfn receptor or fluid phase uptake.
Further, there were no changes in steady state, intracellular
accumulation of the Tfn receptor or lipid recycling despite the
appearance of these morphologically altered early endosomes. Our
results were obtained at a variety of levels of Rab5(Q79L)
overexpression, which caused varying degrees of change in endosome
morphology and after even brief exposure to mutant Rab5 provided little
opportunity for the induction of compensatory mechanisms.
Overexpression of dominant-negative Rab5 mutants (e.g.
Rab5(N133I) or Rab5(S34N)) has been shown by several groups to
inhibit TfnR and fluid phase endocytosis and endosome fusion (5,
8). These mutations block Rab5 function and can exert their
inhibitory effects independently of downstream effectors. By contrast,
the activating mutant studied here, Rab5(Q79L), will require
interaction with downstream effectors to manifest its effects. Cell
type and other variables may determine whether specific downstream
effectors of Rab5 are limiting and therefore whether overexpression of
Rab5(Q79L) will alter the kinetics of membrane trafficking along the
endocytic recycling pathway. Thus, our results do not rule out a
function for Rab5 in controlling membrane trafficking through the
early/sorting endosome.
Regardless, the important findings from these studies are 2-fold.
First, our results clearly establish that Rab5-induced changes in early
endosomal morphology are not predictive of defects in endocytic
membrane trafficking. Second, our results argue that geometric
considerations may contribute to a lesser extent than previously
assumed in determining the bulk sorting and recycling efficiencies of
the early endosomal compartment. Morphometric measurements made of the
tubular and vesicular portions of early endosomes in baby hamster
kidney cells (25) show that 50-70% of total endosomal volume
and 55-90% of total surface area are associated with the tubular
portions of the endosome. Although there is considerable inherent error
in these measurements (25), they also suggest that endosomal geometry
would not be sufficient to account for the observed efficiency of
sorting and recycling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
5 packaging site and 5' to the poly(A) site.
Adenoviruses were generated as previously described (13). Prior to use
in experiments, adenoviruses were plaque-purified to a single viral
population and then amplified.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
enlarged endosomes (upper right).
Expression of HA-Rab5(Q79L) was suppressed when cells were cultured in
the presence of tetracycline (upper middle panel). In all
cases, the endosomes were functional in that fluorescently labeled Tfn
was internalized and delivered to them (Fig. 1B, lower panels). Unexpectedly, examination of the single-round uptake and
recycling kinetics of Tfn revealed that despite formation of enlarged
endosomes there was no perturbation of Tfn uptake in cells induced to
express either WT or mutant Rab5 compared with uninduced control cells
(Fig. 1C). Similarly, there was no change in the steady
state accumulation of Tfn receptors in this endosomal compartment.
These data suggested that the enlarged endosome phenotype is not
predictive of defects in endocytic membrane trafficking.
View larger version (39K):
[in a new window]
Fig. 1.
HA-Rab5(Q79L) expression in stably
transformed tTA-HeLa cells causes enlarged endosomes but does not
effect Tfn endocytosis or recycling. tTA-HeLa cells stably
expressing either HA-Rab5(WT) or HA-Rab5(Q79L) under control of a
tetracycline (tet)-regulatable promoter were clonally
selected as described under "Materials and Methods." Cells were
incubated in the absence (induced) or presence (uninduced) of 1 µg/ml
tetracycline for 48 h. A, immunoblots of cell lysates
probed with antibodies against Rab5 showing expression of endogenous
and HA-tagged Rab5. B, transformed tTA-HeLa cells uninduced
or induced to express either HA-Rab5(WT) or HA-Rab5(Q79L) as indicated
were incubated with Alexa-Tfn (lower panels) for 20 min at
37 °C before fixation, permeabilized in 0.1% saponin, and processed
for indirect immunofluorescence using the 12CA5 anti-HA antibody
(upper panels), as described under "Materials and
Methods." C, single-round kinetics of uptake and recycling
of prebound biotinylated Tfn in tTA-HeLa cells uninduced ( ) or
induced to express either HA-Rab5(WT) (
) or HA-Rab5(Q79L) (
) for
48 h as described under "Materials and Methods." Results
are average ± S.D. of three independent experiments.
View larger version (44K):
[in a new window]
Fig. 2.
High levels of Rab5(Q79L) expression cause
enlargement of endosomes without perturbing endocytic trafficking of
transferrin. tTA-HeLa cells infected with adenoviruses expressing
either HA-Rab5(WT) or HA-Rab5(Q79L) under the control of a
tetracycline-regulatable promoter were incubated for 18 h in the
presence or absence of tetracycline as described under "Materials and
Methods." A, immunoblots of cell lysates probed with
either anti-Rab5 antibodies (right panel) or anti-HA
antibodies (left panel). B, immunofluorescence
images of adenovirally infected tTA-HeLa cells expressing either
HA-Rab5(WT) or HA-Rab5(Q79L) incubated with Alexa-Tfn and
subjected to indirect immunofluorescence with anti-HA monoclonal
antibody as described under "Materials and Methods." Left
panels show HA-Rab5 distribution visualized with a goat anti-mouse
antibody conjugated to Texas Red; right panels show
internalized Alexa-Tfn. C, single-round kinetics of uptake
and recycling of prebound biotinylated Tfn in uninfected tTA-HeLa cells
( ) or in cells infected with either HA-Rab5(WT)-encoding
adenoviruses (
) or HA-Rab5(Q79L)-encoding adenoviruses (
)
assayed 18 h after infection as described under "Materials and
Methods." Inset shows expanded axis for early time points.
Results are average ± S.E. of two independent experiments.
View larger version (68K):
[in a new window]
Fig. 3.
Bolus of Rab5 expression in adenovirally
infected tTA-HeLa cells. Rapid, high level expression of
HA-Rab5(Q79L) in adenovirally infected tTA-HeLa cells was induced after
release from a cycloheximide block as described under "Materials and
Methods." A, immunoblot probed with an anti-Rab5
monoclonal antibody showing rapid induction of Rab5 expression
detectable in 100 µg of whole cell lysates after cycloheximide wash
out for the indicated times. B, adenovirally infected
tTA-HeLa cells induced to express HA-Rab5(WT) or HA-Rab5(Q79L) by
release from cycloheximide block for the indicated times were fixed and
processed for indirect immunofluorescence (IF) using
anti-HA antibody 12CA5. Results shown are representative of at least
three independent experiments.
View larger version (23K):
[in a new window]
Fig. 4.
Endocytic trafficking of transferrin is
unaffected even by rapid induction of high levels of Rab5(Q79L)
expression. Kinetics of uptake and recycling of prebound
biotinylated Tfn were measured in uninfected tTA-HeLa cells
( ), tTA-HeLa cells infected with recombinant adenovirus that
encode HA-Rab5(Q79L) but maintained in the presence of 1 µg/ml
tetracycline (
), or tTA-HeLa cells infected with adenovirus in the
presence of cycloheximide and induced to express either HA-Rab5(WT)
(
) or HA-Rab5(Q79L) (
) after release from cycloheximide for 1, 3, and 12 h as described under "Materials and Methods."
Transferrin endocytosis and recycling were examined using an
enzyme-linked immunosorbent assay-based assay, which monitors the total
level of internalized, prebound, and biotinylated transferrin as
described under "Materials and Methods." Data are plotted as a
percentage of the total biotinylated transferrin initially bound to the
cells. The same data are shown over a shorter time course
(inset).
View larger version (17K):
[in a new window]
Fig. 5.
Overexpression of Rab5(WT) or
Rab5(Q79L) for short or longer periods of time does
not effect fluid phase uptake or lipid recycling. Bolus
expression of HA-Rab5(WT) ( ) or HA-Rab5(Q79L) (
) was induced for
the indicated times in tTA-HeLa cells as in Fig. 3. As controls, cells
were uninfected (
) or infected with HA-Rab5(Q79L) adenovirus and
cultured in the presence of tetracycline (
).
A-C, the kinetics of fluid phase uptake of HRP at
37 °C measured as described under "Materials and Methods" and
expressed in arbitrary units normalized to cellular protein and
relative to control uninfected cells (n = 3, average ± S.E.). D-F, C6-NBD-SM was
internalized for 10 min and surface-associated lipid was removed. Shown
are the kinetics of recycling of internalized lipid during subsequent
incubation at 37 °C.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Yoram Altschuler for assistance with recombinant adenovirus production, Dr. Shana Barbas for construction of HA-tagged Rab5, and Drs. Marino Zerial, Fred Maxfield, and members of the Schmid Laboratory for helpful discussion and/or critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Cancer Institute Grant CA58689 and United States Army Medical Research Acquisition Activity Award DAMD17-99-1-9367.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.
Current address: Dept. of Cell Biology, Oklahoma University Health
Science Center, Oklahoma City, OK 73104.
§ Current address: Dept. Pathologie der Universität Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland.
¶ To whom correspondence should be addressed: Tel.: 858-784-2311; Fax: 858-784-9126; E-mail: slschmid@scripps.edu.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M010387200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PBS, phosphate-buffered saline; WT, wild type; HRP, horseradish peroxidase; Tfn, transferrin; TfnR, transferrin receptor; B-XX-Tfn, biotinylated Tfn; C6-NBD-SM, N-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino) hexanoyl)-sphingosyl phosphocholine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12, 575-625[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Mukherjee, S.,
Ghosh, R. N.,
and Maxfield, F. R.
(1997)
Physiol. Rev.
77,
759-803 |
3. | Gruenberg, J., and Maxfield, F. R. (1995) Curr. Opin. Cell Biol. 7, 552-563[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Barbieri, M. A.,
Li, G.,
Colombo, M. I.,
and Stahl, P. D.
(1994)
J. Biol. Chem.
269,
18720-18722 |
5. | Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, B., and Zerial, M. (1992) Cell 70, 715-728[Medline] [Order article via Infotrieve] |
6. | Gorvel, J.-P., Chavrier, P., Zerial, M., and Gruenberg, J. (1991) Cell 64, 915-925[Medline] [Order article via Infotrieve] |
7. |
Li, G.,
and Stahl, P. D.
(1993)
J. Biol. Chem.
268,
24475-24480 |
8. | Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lutcke, A., Gruenberg, J., and Zerial, M. (1994) EMBO J. 13, 1287-1296[Abstract] |
9. |
Sönnichsen, B.,
DeRenzis, S.,
Nielsen, E.,
Rietdorf, J.,
and Zerial, M.
(2000)
J. Cell Biol.
149,
901-913 |
10. | Stenmark, H., Valencia, A., Martinez, O., Ullrich, O., Goud, B., and Zerial, M. (1994) EMBO J. 13, 575-583[Abstract] |
11. | Damke, H., Gossen, M., Freundlieb, S., Bujard, H., and Schmid, S. L. (1995) Methods Enzymol. 157, 209-220 |
12. | Ghosh, R. N., and Maxfield, F. R. (1995) J. Cell Biol. 128, 549-561[Abstract] |
13. | Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y., and Phipps, M. L. (1997) J. Virol. 71, 1842-1849[Abstract] |
14. | van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowiz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553-563[Abstract] |
15. | Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) J. Cell Biol. 127, 915-934[Abstract] |
16. | Marsh, M., Schmid, S., Kern, H., Harms, E., Male, P., Mellman, I., and Helenius, A. (1987) J. Cell Biol. 104, 875-886[Abstract] |
17. |
Hao, M.,
and Maxfield, F. R.
(2000)
J. Biol. Chem.
275,
15279-15286 |
18. | Dautry-Varsat, A., Ciechanover, A., and Lodish, H. F. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2258-2262[Abstract] |
19. | Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269[Abstract] |
20. |
Sheff, D. R.,
Daro, E. A.,
Hull, M.,
and Mellman, I.
(1999)
J. Cell Biol.
145,
123-139 |
21. | Dunn, K. W., McGraw, T. E., and Maxfield, F. R. (1989) J. Cell Biol. 109, 3303-3314[Abstract] |
22. | Stoorvogel, W., Oorschot, V., and Geuze, H. J. (1996) J. Cell Biol. 132, 21-33[Abstract] |
23. |
Presley, J. F.,
Mayor, S.,
McGraw, T. E.,
Dunn, K. W.,
and Maxfield, F. R.
(1997)
J. Biol. Chem.
272,
13929-13936 |
24. |
Roberts, R. L.,
Barbieri, M. A.,
Pryse, K. M.,
Chua, M.,
Morisaki, J. H.,
and Stahl, P. D.
(1999)
J. Cell Sci.
112,
3667-3675 |
25. | Griffiths, G., Back, R., and Marsh, M. (1989) J. Cell Biol. 109, 2703-2720[Abstract] |