RESEARCH ARTICLE
4499
Lysosome proteins are redistributed during
expression of a GTP-hydrolysis-defective rab5a
Jennifer L. Rosenfeld1,4, Robert H. Moore2, K.-Peter Zimmer3, Estrella Alpizar-Foster4, Wenping Dai2,
M. Nader Zarka1 and Brian J. Knoll4,*
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
2Department of Pediatrics, Pulmonary Section, Baylor College of Medicine, Houston, TX 77030, USA
3Klinik und Poliklinik für Kinderheilkunde, Westfälische Wilhelms-Universität, Albert-Schweitzer-Str. 33, D-48149 Münster, Germany
4Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX 77204, USA
*Author for correspondence (e-mail: [email protected])
Accepted 13 September 2001
Journal of Cell Science 114, 4499-4508 (2001) © The Company of Biologists Ltd
SUMMARY
The functioning of the endocytic pathway is influenced by
a distinct set of rab GTPases, including rab5a, which
regulates homotypic fusion of early endosomes. Expression
of a dominant active, GTPase-defective rab5a accelerates
endosome fusion, causing the formation of a greatly
enlarged endocytic compartment. Here we present
evidence that rab5a also regulates trafficking between
endosomes and lysosomes and may play a role in lysosome
biogenesis. The GTPase defective rab5aQ79L mutant was
inducibly expressed as an EGFP fusion in HEK293 cells,
and the distribution of lysosome proteins and endocytic
markers then assessed by deconvolution fluorescence
microscopy. During expression of EGFP-rab5aQ79L, the
lysosome proteins LAMP-1, LAMP-2 and cathepsin D were
found in dilated EGFP-rab5aQ79L-positive vesicles, which
also rapidly labeled with transferrin Texas Red. Exogenous
tracers that normally traffic to lysosomes after prolonged
chase (dextran Texas Red and DiI-LDL) also accumulated
in these vesicles. Dextran Texas Red preloaded into
lysosomes localized with subsequently expressed EGFPrab5a Q79L, suggesting the existence of lysosome to
endosome traffic. Cells expressing EGFP-rab5a wt or the
dominant negative EGFP-rab5aS34N did not exhibit these
abnormalities. Despite the dramatic alterations in lysosome
protein distribution caused by expression of EGFP-rab5a
Q79L, there was little change in the endocytosis or
recycling of a cell-surface receptor (β2-adrenergic
receptor). However, there was a deficiency of dense βhexosaminidase-containing lysosomes in cells expressing
EGFP-rab5aQ79L, as assessed by Percoll gradient
fractionation. These results suggest that expression of a
GTPase-defective rab5a affects lysosome biogenesis by
alteration of traffic between lysosomes and endosomes.
INTRODUCTION
Golgi network in at least two general ways (Hunziker and
Geuze, 1996). Lumenal hydrolases bind mannose 6-phosphate
receptors (M6PRs) in the trans-Golgi apparatus and are
shuttled to the late endosome compartment for eventual
transport to lysosomes (Brown et al., 1986; Duncan and
Kornfeld, 1988). Alternatively, lysosome membrane proteins
may first transit to the cell surface, then internalize into early
endosomes and subsequently traffic to lysosomes (LippincottSchwartz and Fambrough, 1986). This second itinerary may
also involve trafficking of proteins from the TGN to early
endosomes (Ludwig et al., 1991; Nielsen et al., 2000; Press et
al., 1998), then subsequent recycling to the plasma membrane
(Hunziker and Geuze, 1996). Thus, while many proteins are
enriched in lysosomes, this usually is a dynamic steady-state
condition resulting from complex traffic amongst several
cellular compartments.
Endocytic trafficking events are in part governed by rasrelated GTPases of the rab family. Rab proteins are 23-25 kDa
in mass and tightly bound to membranes via C-terminal
geranylgeranyl modifications (Novick and Zerial, 1997). Each
rab isoform appears to be associated with a specific subcellular
compartment, and some rabs are known to regulate traffic in
the endosome-lysosome system. Transport to late endosomes
Organelles within the endosome/lysosome system play
important roles in cellular physiology. The uptake of
extracellular ligands and their transport to lysosomes has been
extensively studied (Goldstein et al., 1985); however, lysosome
function is critical for many other cellular processes. The
termination of signaling by cell-surface receptors, such as
receptor tyrosine kinases (Stoscheck and Carpenter, 1984) and
G-protein-coupled receptors (Moore et al., 1999b; Trejo et al.,
1998) can occur by endocytosis and subsequent receptor
degradation in lysosomes. In cells of the immune system,
antigen presentation may involve degradation of internalized
antigens by lysosomal proteases, generating peptides that bind
MHC class II molecules (Nakagawa and Rudensky, 1999).
Alterations in the endosome-lysosome system are associated
with some forms of Alzheimer’s disease and Down’s
syndrome, possibly contributing to changes in the production
and/or secretion of amyloidogenic protein by neurons (Cataldo
et al., 1996; Cataldo et al., 2000).
Considerable effort has been given to understanding the
biogenesis of lysosomes in mammalian cells. Proteins regarded
as residents of lysosomes can be directed there from the trans-
Key words: Rab5a, Lysosome, Endosome, β-hexosaminidase, β2adrenergic receptor
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JOURNAL OF CELL SCIENCE 114 (24)
from the trans-Golgi network of lumenal lysosome proteins
liganded to M6PRs is regulated by rab9 (Lombardi et al.,
1993). Early events in endocytosis are regulated by rab5a
(Bucci et al., 1992), while trafficking from sorting endosomes
to lysosomes is governed by rab7 (Feng et al., 1995;
Mukhopadhyay et al., 1997; Press et al., 1998; Vitelli et al.,
1997). The delivery of transferrin receptors from sorting
endosomes to perinuclear recycling endosomes appears to be
regulated by both rab11 and rab5a (Ren et al., 1998; Ullrich et
al., 1996; Wilcke et al., 2000).
Rab5a is a key regulator of endocytosis because it is rate
limiting for homotypic endosome fusion (Bucci et al., 1992;
Gorvel et al., 1991). Rab5a mutants defective in guaninenucleotide binding (rab5a S34N or rab5a N133I) are dominant
negative in action, and when expressed in transfected cells,
inhibit endosome fusion and cause the accumulation of small
vesicles at the cell periphery (Bucci et al., 1992). By contrast,
the GTPase defective mutant rab5aQ79L is dominant active
and causes the formation of greatly enlarged endosomes due
to a stimulation of endosome fusion (Stenmark et al., 1994).
Overexpression of rab5a wild-type (wt) or rab5aQ79L
increases the steady-state accumulation of fluid phase
endocytic markers (Li and Stahl, 1993); however, the rate of
receptor endocytosis itself may be unaltered (Ceresa et al.,
2001; Seachrist et al., 2000). Various lines of evidence suggest
that while rab5a-GTP is necessary for endosome fusion,
the GTPase activity of rab5a is not (Barbieri et al., 1994;
Hoffenberg et al., 1995a; Rybin et al., 1996; Stenmark et al.,
1994). It has been proposed that the function of the GTPase
activity is to maintain a dynamic equilibrium between rab5aGDP and rab5a-GTP (Rybin et al., 1996) that can be regulated
by GTPase activators (Lanzetti et al., 2000) and proteins that
stimulate guanine-nucleotide exchange (Hoffenberg et al.,
2000; Horiuchi et al., 1997). Evidence from studies of rab5a
(McBride et al., 1999), rab1 (Allan et al., 2000) and the yeast
rab-like protein Ypt7 (Ungermann et al., 2000) suggest a
functional role for these small GTPases in the recruitment of
SNARE proteins to membranes to facilitate budding or fusion
reactions.
There is less information available about how rab5a activity
influences the development of lysosomes. Because some
fractions of lysosomal proteins reach their destinations via
endosomes, experimental changes in rab5a activity might be
expected to alter the morphology or function of lysosomes.
Expression of the dominant-negative rab5aS34N mutant
decreases the rate of endocytosis and degradation of epidermal
growth factor receptors (Barbieri et al., 2000; Papini et al.,
1997); however, it is unclear whether endosome to lysosome
transport is affected. In addition, degradation of low-density
lipoprotein (LDL) is greatly reduced by the expression of
rab5aS34N, possibly due to a defect in LDL-receptor
endocytosis (Vitelli et al., 1997). More direct evidence for a
role of rab5a in lysosome function comes from studies of
macrophage cells, where the rate of phagosome maturation is
reduced by antisense inhibition of rab5a activity, while
maturation is accelerated during overexpression of wild-type
rab5a (Alvarez-Dominguez and Stahl, 1999). Interestingly,
expression of a dominant active rab5a in MDCK cells caused
the formation of enlarged rab7-positive vesicles (D’Arrigo et
al., 1997), suggesting a role for rab5a downstream of early
endosomes.
To further explore the role of rab5a in mammalian lysosome
biogenesis, we expressed rab5a as a fusion with enhanced
green fluorescent protein (EGFP) using an inducible
expression system in cultured cells, and looked for changes in
the distribution of lysosome proteins and several endocytic
tracers. Surprisingly, modest expression of the GTPasedeficient EGFP-rab5aQ79L caused an extensive redistribution
of lysosome proteins into large vesicles that also contain
EGFP-rab5aQ79L, without an appreciable effect on the
endocytosis or recycling of a cell surface receptor. These
findings support the idea that rab5a GTPase activity plays a
role in the formation of lysosomes.
MATERIALS AND METHODS
Cells and reagents
EcR293 cells were obtained from Invitrogen (Carlsbad, CA) and
cultured in Dulbecco’s modified Eagle medium (DMEM) with 10%
fetal bovine serum and 200 µg/ml Zeocin (Invitrogen). Antibody
sources were as follows: monoclonal anti-bovine cation-independent
mannose-6-phosphate receptor (CI-M6PR) antibody 22D4 (a gift of
J. Rohrer, F. Miescher Institute); monoclonal anti-LAMP-1 antibody
CD107A (Research Diagnostics Inc., Flanders, NJ); monoclonal antiLAMP-2 antibody CD107B (Pharmingen, San Diego, CA); rabbit
polyclonal anti-cathepsin D (Calbiochem, San Diego, CA);
monoclonal anti-rab5a antibody, clone 15 (BD Transduction Labs,
San Diego, CA). Fluorescent secondary antibodies (goat anti-rabbit
IgG and goat anti-mouse IgG), DiI-LDL, DQ Red BSA and dextran
Texas Red (10,000 Mr) were purchased from Molecular Probes
(Eugene, OR). Ponasterone was obtained from Invitrogen, and
Hygromycin B and FuGene 6 transfection reagent were purchased
from Roche Molecular Biochemicals. All other reagents were from
Sigma Chemical Co. (St Louis, MO) unless otherwise noted.
DNA constructs
A rab5a-EGFP fusion was created by subcloning a rab5a wt cDNA
fragment (Hoffenberg et al., 1995b) into pEGFP-C1 (Clontech,
Palo Alto, CA). pEGFP-rab5a wt was mutagenized using the
QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla,
CA) with the primer 5′-GGATACAGCTGGCCTAGAACGATACCATAG-3′ to create the rab5aQ79L mutant and the primer 5′-GTCCGCTGTTGGTAAAAATAGCCTAGTGCTTC-3′ to create the
rab5aS34N mutant. These EGFP-rab5a fusions were then subcloned
into the vector pIND-Hygro (Invitrogen) for inducible expression
in EcR293 cells, and into pcDNA3.1 (Invitrogen) for transient
transfections.
Inducible expression of EGFP-rab5a
The EcR293 line was derived from HEK293 and expresses the
regulatory protein VgRXR, a chimeric steroid receptor that is
activated by synthetic ecdysteriods such as ponasterone (No et al.,
1996). The vector pIND-Hygro has a promoter element responsive to
VgRXR, cloning sites for insertion of open reading frames and a
hygromycin B resistance gene. pIND-Hygro/EGFP-rab5a wt, pINDHygro/EGFP-rab5aQ79L and pIND-Hygro/EGFP-rab5aS34N were
transfected into EcR293 cells using FuGENE6, and clones resistant
to hygromycin B (200 µg/ml) were screened for ponasteroneinducible fluorescence. For the induction of expression, cells were
treated for up to 96 hours with 5 µM ponasterone, while control cells
were treated with vehicle alone (0.125% ethanol). The fraction of cells
expressing EGFP-rab5a in the presence of ponasterone was 90-95%.
Immunoblotting
Transfected cells were washed with PBS and then quickly dissolved
in Laemmli sample buffer (Laemmli, 1970). Samples of 20 µg total
Rab5a and trafficking to lysosomes
protein were electrophoresed through SDS-PAGE and transferred to
Immobilon-P membranes (Millipore, Bedford, MA). The membranes
were probed with anti-rab5 monoclonal antibodies at a dilution of
1:500 and bound antibodies were detected by chemiluminescence
(Pierce Chemical Co., Rockford, IL). Protein bands visualized on Xray films were quantified by densitometry using SigmaGel 1.0 (SPSS
Science, Chicago, IL).
Uptake of endocytic tracers
For labeling with transferrin Texas Red, the cells were washed and
incubated in serum-free medium for 30 minutes, pulsed with 20 µg/ml
of the tracer for 5 minutes, then rapidly chilled and fixed. For pulsechase analysis of fluid phase uptakes, dextran Texas Red (1 mg/ml)
was added directly to the medium for 1 hour, then the cells were
washed and incubated in complete medium for an additional 6 hours
before washing and fixation. To assess lysosome to endosome traffic,
cells were incubated with dextran Texas Red for 1 hour, then washed
and chased for 6 hours, and finally incubated with ponasterone for 72
hours prior to fixation. For LDL uptakes, the cells were cultured in
the presence of ponasterone or vehicle for 72 hours in medium
containing 10% NuSerum (Life Technologies) in place of fetal bovine
serum, then Di-LDL was added to 5 µg/ml for 5 minutes. The labeled
medium was removed, the cells washed and then incubated in
complete medium for a further 1 hour prior to fixation.
Immunofluorescence microscopy
Cells growing on glass coverslips in 6-well clusters were washed in
PBS with 1.2% sucrose (PBSS), fixed with 4% paraformaldehyde in
PBSS at 4°C for 10 minutes, and then washed again with PBSS. The
following steps were done at room temperature, with PBSS used for
washes. The coverslips were incubated in 0.34% L-lysine, 0.05% Nam-periodate for 20 minutes and permeabilized with 0.2% Triton X100. The cells were then blocked with 10% normal goat serum (NGS)
for 15 minutes. Primary antibodies, diluted in PBSS with 0.2% NGS
and 0.05% Triton X-100, were added to the cells and left for 1 hour.
The coverslips were washed four times before incubation with
secondary antibodies using the same procedure as for the primary
antibodies. We used the following concentrations of antibodies: anticathepsin D, 20 µg/ml; anti-LAMP-1, 5 µg/ml; anti-LAMP-2, 5
µg/ml; anti-CI-M6PR, 1:200; secondary antibodies, 1:100 dilution.
The coverslips were mounted in Mowiol and viewed using a
DeltaVision deconvolution microscopy system (Applied Precision
Inc., Issaquah, WA) in the Baylor College of Medicine Integrated
Microscopy Core. Optical sections of 150 nm (10-15 in number) were
obtained and deconvolved, and then five sections through the cell
center were combined to produce the images shown.
Immunoelectron microscopy
Sectioning and labeling of ultrathin frozen sections (50 nm) of EGFPrab5aQ79L expressing cells using the technique of Tokuyasu were
performed as described in detail elsewhere (Zimmer et al., 1998).
Small specimens were cryoprotected by polyvinylpyrrolidone/
sucrose, frozen in liquid nitrogen and sectioned with a
cryoultramicrotome (LEICA EM Ultracut R FCS) at –100 to –110°C.
Thawed sections were incubated at room temperature with the
monoclonal antibody against GFP (Scompany, dilution of 1:10) for
45 minutes and goat anti-mouse IgG conjugated with 6 nm gold
(Dianova, D-Hamburg, dilution of 1:10). Double-labeling was
performed by incubating the sections with a polyclonal antibody
against LAMP-2 (a gift of M. Fukuda, San Diego, dilution of 1:50)
and goat anti-mouse IgG conjugated with 12 nm gold (Dianova,
dilution of 1:50). After labeling, the grids were contrasted, embedded
in 2% methylcellulose and examined in a Philips 400 electron
microscope (Kassel, Germany).
Measurement of β2AR endocytosis and recycling kinetics
Cells growing in 24-well clusters were treated with 5 µM ponasterone
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or vehicle alone (0.125% ethanol) for 72 hours. Isoproterenol (10 µM)
was added to the cells in triplicate wells for varying times up to 20
minutes, then the wells were aspirated and washed with ice-cold
serum-free medium containing 20 mM Hepes pH 7.4 (DMEM-H).
Cell surface receptors were quantified by incubation at 4°C for 90
minutes with 6 nM [3H]CGP12177, a hydrophilic radioligand that
selectively binds surface β2ARs (Staehelin and Hertel, 1992). The
cells were washed twice with cold DMEM-H, then lysed in the wells
with 0.1% SDS, 0.1% NP-40 and the lysates counted by scintillation
spectroscopy. Nonspecific binding was determined by incubations
with 3 µM propranol, and was always less than 5%. The fraction of
receptors left on the cell surface was plotted versus time of agonist
exposure, and the curves fitted by nonlinear regression using the
program GraphPad Prism (v. 3). The rate of approach to a steady-state
level of surface and internal receptors is determined by the first-order
rate constants for receptor endocytosis (ke) and recycling (kr). Unique
values for these rate constants were estimated by curve-fitting to
equation 4 in Morrison et al. (Morrison et al., 1996).
Percoll density gradients
Stably transfected cells inducibly expressing EGFP-rab5aQ79L were
grown in the presence of 5 µM ponasterone or vehicle alone for 72
hours. The cells were washed with PBS and then suspended in 1×
SHE, which contained 0.25 M sucrose, 10 mM Hepes, pH 7.42, 2 mM
EDTA, 1× Complete® proteinase inhibitor (Roche Biomolecular). The
cell suspension was homogenized by repeated passage through a 25G
needle, and then centrifuged at 500 g to obtain a post-nuclear
supernatant. This was overlayed onto a solution of 25% Percoll in 1×
SHE with 25 mM ATP and centrifuged at 36,000 g for 65 minutes in
Fig. 1. (A) Immunoblot analysis of EGFP-rab5aQ79L induction. An
EcR293 cell line stably transfected with pIND-Hygro/EGFPrab5aQ79L (EcR293 pIND-Hygro/EGFP-rab5aQ79L) was treated
for 24, 48, 72 or 96 hours with 5 µM ponasterone or with vehicle
(veh: 0.125% ethanol) for 96 hours only. Total cell lysates were
electrophoresed through SDS-PAGE, blotted and probed with a
monoclonal anti-rab5 antibody. Endogenous rab5 migrates at
approximately 25 kDa, and EGFP-rab5aQ79L at 50 kDa.
(B) Deconvolution fluorescence microscopy of EcR293/EGFPrab5aQ79L cells treated for 72 hours with 5 µM ponasterone (pon) or
vehicle (veh).
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Fig. 2. Rapid labeling of EGFPrab5a-containing vesicles with
transferrin Texas Red. EcR293
pIND-Hygro/EGFP-rab5a wt cells
(A) and EcR293 pINDHygro/EGFP-rab5aQ79L cells
(B) were grown for 72 hours in the
presence of 5 µM ponasterone,
then incubated for 5 minutes with
transferrin Texas Red (20 µg/ml)
prior to rapid fixation in 4%
paraformaldehyde. The cells were
imaged by deconvolution
fluorescence microscopy. Bar, 10
µm.
a 70.1 Ti rotor at 4°C. Immediately after collection of fractions, 25 µl
aliquots were assayed for β-hexosaminidase activity by incubation
with 0.3 mM methylumbelliferone in acetate buffer (100 mM sodium
acetate and 0.1% Triton X-100) at 37°C for 1 hour in the dark.
Trichloroacetic acid was added to a final concentration of 10% to stop
the reactions. Samples were diluted 1:20 with 0.5 M glycine and 0.5
M sodium carbonate buffer, added in triplicate to white U-bottom 96well trays, then read at an excitation of 365 nm and emission of 450
nm on a Dynatech Fluorolite 100.
RESULTS
Inducible expression of EGFP-rab5a in HEK293 cells
The ecdysone inducible mammalian expression system was
used to control the levels of EGFP-rab5a in EcR293 cells, as
described in Materials and Methods. Immunoblot analysis of
one EcR293 pIND-Hygro/EGFP-rab5aQ79L clone is shown in
Fig. 1A. Incubation in the presence of 5 µM ponasterone for
up to 72 hours induced expression of EGFP-rab5aQ79L to an
average extent of 3.4-fold over endogenous rab5a, with no
detectable expression in uninduced cells, as assessed by
densitometry. Similar results were obtained with cells
inducibly expressing EGFP-rab5a wt and EGFP-rab5a S34N
(data not shown). Cells incubated with vehicle alone showed
very little fluorescence of EGFP-rab5aQ79L (Fig. 1B, left), in
agreement with the immunoblotting results. By contrast, cells
incubated with 5 µM ponasterone for 72 hours showed a
pronounced expression of EGFP-rab5aQ79L, as well as the
characteristic dilated endosome morphology normally found
with expression of this mutant rab protein (Fig. 1B, right).
Within 5 minutes of adding transferrin Texas Red to the
medium, both EGFP-rab5a wt (Fig. 2A) and EGFP-rab5aQ79L
(Fig. 2B) containing endosomes were mostly positive for label.
These results are in agreement with similar studies performed
previously with BHK-21 and Hela cells (Stenmark et al.,
1994).
Lysosome proteins localize with EGFP-rab5aQ79L
We next labeled induced and control cells with antibodies
Fig. 3. Lysosome/late endosome proteins localize with EGFPrab5aQ79L. EcR293 pIND-Hygro/EGFP-rab5aQ79L cells were
treated with vehicle (left) or 5 µM ponasterone (right) for 72 hours
and then fixed and labeled with antibodies against LAMP-1, LAMP2, cathepsin D or CI-M6PR as indicated. Secondary antibodies were
TRITC (red) goat anti-mouse or goat anti-rabbit IgG. The labeled
cells were imaged by deconvolution fluorescence microscopy. Bar,
10 µm.
against several well-characterized lysosome proteins.
Lysosome-associated membrane proteins LAMP-1 and
LAMP-2 reach lysosomes by first sorting from the trans-Golgi
network to early endosomes, either directly (Hunziker and
Geuze, 1996) or via the plasma membrane (Akasaki et al.,
1996; Akasaki et al., 1995). In cells expressing EGFPrab5aQ79L, both LAMP-1 and LAMP-2 localized extensively
with EGFP-rab5aQ79L containing vesicles (Fig. 3, right).
Interestingly, a considerable fraction of LAMP-1 and LAMP2 appeared within dilated EGFP-rab5aQ79L-bounded vesicles,
giving the appearance of multivesicular bodies (Fig. 3, right).
Another lysosome protein, cathepsin D, is a lumenal enzyme
that traffics to lysosomes as a ligand of the mannose 6phosphate receptor (M6PR) (Kornfeld, 1992). Antibodies to
cathepsin D also labeled the enlarged EGFP-rab5aQ79L-
Rab5a and trafficking to lysosomes
4503
Fig. 4. Immunogold labeling and electron
microscopy of EGFP-rab5aQ79L-expressing
cells. EcR293pIND-Hygro /EGFP-rab5aQ79L
cells were grown in the presence of ponasterone
for 72 hours, then fixed in 4% PFA and
processed for immunogold labeling and electron
microscopy as described in Materials and
Methods. EGFP, 6 nm gold (arrowheads);
LAMP-2, 12 nm gold (arrows); M,
mitochondrion. Magnification, 65,000×.
containing vesicles (Fig. 3, right), primarily labeling the lumen,
with outer membrane labeling being much less apparent than
for LAMP-1 or LAMP-2. Consistent with this finding, EGFPrab5aQ79L-containing endosomes also labeled with DQ Red
BSA, a fluid-phase marker whose fluorescence is dequenched
in the presence of proteases (Reis et al., 1998) (data not
shown). The colocalization of cathepsin D and EGFPrab5aQ79L suggested that the distribution of M6PRs might
also be effected. At steady-state, most CI-M6PR shows a
perinuclear localization, and to some degree this is apparent in
vehicle-treated cells (Fig. 3, left). In induced cells, the
perinuclear distribution of CI-M6PR was less apparent and a
fraction of receptors localized adjacent to or within EGFPrab5aQ79L containing vesicles (Fig. 3, right).
Vesicles containing both EGFP-rab5aQ79L and LAMP-1 or
LAMP-2 had a multivesicular appearance at the level of light
microscopy (Fig. 3). To examine this more closely, cells
expressing EGFP-rab5aQ79L were processed for immunogold
electron microscopy using antibodies against EGFP and
LAMP-2. A representative image (Fig. 4) shows a greatly
enlarged endosome with labeling for EGFP (arrowheads) that
is also positive for LAMP-2 (arrows). These endosomes
contained numerous internal vesicles, characteristic of
mutlivesicular bodies, or prelysosomal structures.
In order to determine whether the effect on lysosomal
protein distribution of EGFP-rab5aQ79L can be attributed to
its lack of GTPase activity, cells expressing EGFP-rab5a wt or
the dominant negative mutant EGFP-rab5a S34N were labeled
with antibody to LAMP-1. This lysosome protein did not
localize with EGFP-rab5a wt (Fig. 5A), in agreement with a
previously published finding (Bucci et al., 2000). The
distribution of the dominant negative mutant EGFP-rab5aS34N
was diffuse and to some degree localized to a perinuclear
region; however, there was no apparent localization with
LAMP-1 (Fig. 5B). This finding supports the view that the
GTPase deficiency of EGFP-rab5aQ79L mutant is causing a
mis-sorting of lysosome proteins.
Although expression of EGFP-rab5aQ79L is barely
detectable in the stable inducible cell line, it is conceivable that
this may be sufficient to cause adaptive changes in the
transfected cells. To rule this out, HEK293 cells were transiently
transfected for 48 hours with pcDNA3.1/EGFP-rab5aQ79L or
pcDNA3.1/EGFP-rab5a wt, then labeled with antibody against
LAMP-1 and imaged as described above. The distribution of
LAMP-1 in transiently transfected cells was very similar to that
observed in the inducible cell lines (data not shown).
Fig. 5. LAMP-1 distribution in
cells expressing EGFP-rab5a wt
or EGFP-rab5aS34N.
EcR293pIND-Hygro /EGFPrab5a wt cells (A) and
EcR293pIND-Hygro/EGFPrab5a S34N cells (B) were
induced for 72 hours with 5 µM
ponasterone, then fixed and
labeled with anti-LAMP-1
primary and TRITC (red)
secondary antibodies. The
labeled cells were imaged by
deconvolution fluorescence
microscopy. Bar, 10 µm.
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JOURNAL OF CELL SCIENCE 114 (24)
Fig. 6. Endocytic markers accumulate in EGFP-rab5aQ79Lcontaining vesicles. (A,B). EcR293pIND-Hygro /EGFP-rab5a wt
cells (A) and EcR293pIND-Hygro/EGFP-rab5aQ79L cells (B) were
induced with 5 µM ponasterone for 72 hours, then pulse labeled for 1
hour with dextran Texas Red, followed by a 6 hour chase prior to
fixation. (C,D). EcR293 pIND-Hygro/EGFP-rab5a wt cells (C) and
EcR293 pIND-Hygro/EGFP-rab5aQ79L cells (D) were induced with
5 µM ponasterone for 72 hours, then pulse labeled for 5 minutes with
Di-LDL, followed by a 1 hour chase prior to fixation. The labeled
cells were imaged by deconvolution fluorescence microscopy. Bar,
10 µm.
Endocytic tracers accumulate in EGFP rab5aQ79L
containing endosomes
The proteins so far examined traffic to lysosomes by various
pathways that are believed to originate at the trans-Golgi
network. To determine specifically whether traffic between the
plasma membrane and lysosomes is affected by expression
of EGFP-rab5aQ79L, cells were labeled with fluorescent
endocytic tracers that are known to traffic from endosomes to
lysosomes, then subjected to imaging. Dextran Texas Red was
fed to cells for 1 hour, then washed and chased for 6 hours prior
to fixation and imaging. In cells expressing EGFP-rab5a wt,
dextran Texas Red was chased from endosomes into punctate
vesicles that showed no overlap with EGFP-rab5a wt (Fig. 6A).
By contrast, a considerable proportion of dextran Texas Red
remained within the lumen of enlarged EGFP-rab5aQ79L
containing vesicles after chase (Fig. 6B). Similar results were
obtained using BSA Texas Red (not shown). To determine
whether trapping of endocytic tracer within endosomes by
expression of EGFP-rab5aQ79L was limited to fluid-phase
proteins, DiI-LDL was added to cells for 5 minutes, then
removed and chased for 1 hour. In cells expressing EGFPrab5a wt, internalized label sorted from endosomes into
discrete, punctate vesicles (Fig. 6C). However, in cells
expressing EGFP-rab5aQ79L, the majority of Di-LDL
remained within endosomes after a 1 hour chase (Fig. 6D).
Rates of endocytosis and recycling during EGFPrab5aQ79L expression
Our results indicate that proteins targeted to lysosomes can
accumulate in EGFP-rab5aQ79L-containing endosomes. One
Fig. 7. Endocytosis and recycling of a cell surface receptor. EcR293
pIND-Hygro/EGFP-rab5aQ79L cells were stably transfected with
pcDNA3.1/β2AR, then incubated with either vehicle (䊏) or 5 µM
ponasterone (䊐) for 72 hours. The cells were treated with 10 µM
isoproterenol for varying times, then chilled, washed and kept on ice
with 6 nM [3H]CGP12177 for 90 minutes to selectively label surface
receptors. The fraction of receptors left on the surface was plotted
versus time of agonist exposure, and the curves modeled as described
(Morrison et al., 1996) to obtain estimates for the first-order rate
constants of endocytosis (ke) and recycling (kr). The rate constant ke
for control cells was 0.199±0.010 minutes–1 and for ponasteronetreated cells, 0.151±0.005 minutes–1 (significantly different,
P=0.029). The rate constant kr was 0.053±0.005 minutes–1, and for
ponasterone-treated cells, 0.038±0.010 minutes–1 (not significant,
P=0.1005) (n=3).
possible explanation is that in HEK293 cells, EGFPrab5aQ79L causes an inhibition in recycling from endosomes
to the plasma membrane, or affects some other generalized
change in endocytosis or recycling. Studies using HeLa cells
have shown that expression of rab5aQ79L has no appreciable
effect on transferrin receptor endocytosis or recycling (Ceresa
et al., 2001) despite the presence of greatly enlarged
endosomes. In HEK293 cells, the endocytosis of cell-surface
β2-adrenergic receptors (β2ARs) in transient co-transfections
with rab5aQ79L appears unaffected, although the recycling
rate was not measured (Seachrist et al., 2000). To determine
whether EGFP-rab5aQ79L expression in our inducible
HEK293 cells alters receptor endocytosis or recycling, the line
was stably transfected to express human β2ARs, then the rate
of receptor internalization was measured in response to βagonist treatment. In the presence of ponasterone, this cell line
exhibited the same dilated endosomes as the parent line, and
internalized β2ARs localized to these endosomes together with
lysosome markers (data not shown). The internalization of
β2ARs was then examined by measuring the loss of surface
receptors as a function of time after adding β-agonist (Fig. 7).
Internalization curves were modeled to derive first-order rate
constants for both endocytosis (ke) and recycling (kr) (Morrison
et al., 1996). During the relatively short time courses used to
measure these rates, the predominant mode of β2AR trafficking
is between early endosomes and the plasma membrane (Moore
et al., 1999a; Moore et al., 1999b). While EGFP-rab5aQ79L
expression appeared to cause a slight inhibition in the rate of
β2AR endocytosis, there was no significant difference in
recycling compared with control cells (Fig. 7 legend). The
recycling rate of internalized receptors can also be determined
directly by measuring receptor reappearance at the cell surface
following the removal of agonist after steady-state receptor
internalization (Morrison et al., 1996). The recycling rates
Rab5a and trafficking to lysosomes
4505
Fig. 9. Distribution of β-hexosaminidase in Percoll gradients.
EcR293 pIND-Hygro/EGFP-rab5aQ79L cells were treated with
vehicle (䊏) or ponasterone (䊐) for 72 hours, then homogenized and
applied to self-forming Percoll gradients (see Materials and
Methods). After centrifugation, samples were taken from the bottom
(fraction 1), and aliquots assayed for β-hexosaminidase activity and
protein concentration. Results are representative of three independent
experiments.
localization. It is possible that the prolonged culture of cells
required for this protocol contributes to this apparent
cytoplasmic localization. By contrast, cells expressing EGFPrab5aQ79L showed large fluorescent vesicles containing Texas
Red marker (Fig. 8B). Labeling of EGFP-rab5aQ79Ltransfected cells with dextran Texas Red in the absence of
inducing agent showed a punctate labeling pattern more typical
of lysosomes (Fig. 8C).
Fig. 8. Dextran Texas Red preloaded into lysosomes localizes with
EGFP-rab5aQ79L. Cells were incubated with dextran Texas Red (1
mg/ml) for 1 hour, washed and then chased for 6 hours in the
absence of tracer. The cells were then induced with 5 µM
ponasterone or vehicle for 72 hours prior to fixation, and imaged by
deconvolution fluorescence microscopy. (A) EcR293 pINDHygro/EGFP-rab5a wt; (B) EcR293 pIND-Hygro/EGFP-rab5aQ79L;
(C) EcR293 pIND-Hygro/EGFP-rab5aQ79L pulse labeled with
dextran Texas Red and incubated 72 hours with vehicle alone. Bar,
10 µm.
β-hexosaminidase distribution in cells expressing
EGFP-rab5aQ79L
The aberrant distribution of lysosome proteins in cells
expressing EGFP-rab5aQ79L suggested that there might be a
defect in the formation of dense lysosomes. Percoll gradient
fractionation was used to assess the distribution of the
lysosome enzyme β-hexosaminidase in EGFP-rab5aQ79Lexpressing cells and in uninduced controls. In fractions from
vehicle-treated cells, most of the β-hexosaminidase-specific
activity was seen in a peak near the bottom of the gradient,
while such a peak was absent or greatly reduced in cells
expressing EGFP-rab5aQ79L (Fig. 9).
DISCUSSION
determined this way were similar in both the presence and
absence of ponasterone (data not shown).
Tracer from preloaded lysosomes localizes with
EGFP-rab5aQ79L
In addition to altering the traffic of proteins from early
endosomes to lysosomes, we wondered if movement of
materials from lysosomes to endosomes might occur during
expression of EGFP-rab5aQ79L. To test this possibility, cells
were loaded with dextran Texas Red, washed and chased for 6
hours, then induced with ponasterone for 72 hours. Cells
expressing EGFP-rab5a wt showed no overlap between EGFP
and Texas Red (Fig. 8A), although the distribution of EGFPrab5a wt was somewhat diffused, suggesting some cytoplasmic
We present evidence that a GTPase defective rab5a interferes
with normal lysosome biogenesis in mammalian cells.
Expression of EGFP-rab5aQ79L caused the formation of large
vesicles containing integral membrane lysosome proteins
(LAMP-1 and LAMP-2), a lumenal protein that is normally a
ligand of M6PRs (cathepsin D), and CI-M6PRs (Fig. 3; Fig.
4). Endocytic tracers remained within EGFP-rab5aQ79Lcontaining vesicles even after prolonged chase, in contrast to
cells expressing EGFP-rab5a wt, where such tracers normally
sort to separate compartments (Fig. 6). Moreover, material
preloaded into lysosomes was found within endosomes after
subsequent expression of EGFP-rab5aQ79L (Fig. 8) and there
was a pronounced deficiency in β-hexosaminidase-containing
4506
JOURNAL OF CELL SCIENCE 114 (24)
dense lysosomes (Fig. 9). However, trafficking between the
plasma membrane and early endosomes appeared to occur
normally (Fig. 7). These results suggest that specific changes
in the trafficking of proteins between endosomes and
lysosomes are caused by expression of rab5aQ79L.
The remarkable redistribution of lysosome proteins to
EGFP-rab5aQ79L-containing vesicles is reminiscent of what
occurs when cells are treated with chloroquine to block
endosome-lysosome transport (Lippincott-Schwartz and
Fambrough, 1987). This similarity suggests that EGFPrab5aQ79L inhibits such transport events, causing the
accumulation within endosomes of lysosome proteins that
normally traverse the endosome compartment before reaching
lysosomes. This interpretation would be consistent with
findings that expression of rab5aQ79L inhibits transport (of
transferrin) from early endosomes to perinuclear recycling
endosomes (Ullrich et al., 1996), inhibits degradation of LDL
and epidermal growth factor (McCaffrey et al., 2001) and
inhibits to some degree the degradation of ricin in MDCK
cells (D’Arrigo et al., 1997). In that study, abnormalities
in late endosomes/lysosomes was further suggested by the
observation of enlarged rab7-positive vesicles in cells
expressing myc-tagged rab5aQ79L, and a partial
colocalization of these two proteins. We observed a similar
morphology in HEK293 cells expressing GFP-rab7 and myctagged rab5aQ79L (data not shown). However, the inhibition
of transport from endosomes to lysosomes per se may not be
sufficient to explain our findings. Expression of a dominantnegative syntaxin-7 blocks traffic from early to late
endosomes in NIH3T3 cells, yet does not appear to cause the
accumulation of LAMP-2 or cathepsin D in early endosomes
(Nakamura et al., 2000). Also, while our data suggest that the
expression of EGFP-rab5aQ79L may inhibit trafficking from
early endosomes to lysosomes, there also appears to be a
stimulation of traffic in the opposite direction (Fig. 8).
However, our results do not allow us to distinguish between a
stimulation of direct fusion between lysosome and endosomes
versus a stimulation of vesicular trafficking from lysosome to
endosomes.
Recent evidence suggests that rab7 also plays an important
role in lysosome biogenesis. Expression of GTPase-defective
rab7 in HeLa cells increases the size of lysosomes and
the extent of perinuclear aggregation, while expression of
dominant negative rab7 causes dispersion of lysosomes
throughout the cytoplasm. These aberrant organelles are not
accessible to endocytic tracers; however, there is no apparent
change in the early or late endosome compartments (Bucci et
al., 2000). In BHK-21 cells, dominant negative rab7 expression
causes an increase in the proportion of CI-MPR and cathepsin
D in early endosome compartments. By contrast, lgp 120, a
homologue of LAMP-1, shows a normal distribution under
these conditions (Press et al., 1998). These results suggest that,
in BHK-21 cells, CI-M6PR/cathepsin D is delivered to
lysosomes through the early endosome compartment, whereas
lgp 120 is delivered via late endosomes. The localization of
LAMP-1 and LAMP-2 with EGFP-rab5aQ79L observed in our
study suggests that, in contrast with BHK-21 cells, these
lysosomal proteins traffic to lysosomes via early endosomes in
HEK293 cells. Our results are consistent with what was found
in rat hepatocytes, where significant fractions of both LAMP1 and LAMP-2 are sorted to the plasma membrane and early
endosomes before eventual transport to lysosomes (Akasaki et
al., 1995).
In our experimental system, we do not detect significant
changes in β2AR recycling during EGFP-rab5aQ79L
expression (Fig. 7), suggesting a relatively specific effect on
trafficking of proteins between endosomes and lysosomes.
Previous studies where rab5aQ79L caused changes in
transferrin recycling and endocytosis employed acute 5-10fold overexpression mediated by recombinant vaccinia viruses
over a 4-5-hour period after infection (Stenmark et al., 1994),
rather than the approximately threefold expression relative to
endogenous rab5a after 48-72 hours reported here. Stenmark’s
study also used HeLa or BHK-21 cells, so that differences in
cell type could be significant. Further, subsequent studies of
rab5aQ79L overexpression using recombinant adenoviruses
failed to detect changes in the rates of transferrin recycling or
endocytosis (Ceresa et al., 2001). Our findings suggest that
alteration of intracellular sorting events may be a more
significant consequence of inactivating the rab5a GTPase.
The accumulation of cathepsin D within enlarged rab5acontaining endosomes was recently observed in pyramidal
neurons from patients with sporadic Alzheimer’s disease
(Cataldo et al., 1997). These abnormal endosomes appeared
to be positive for rabaptin 5 and EEA-1, proteins that bind
rab5a-GTP specifically, suggesting enhanced rab5a activation
(Cataldo et al., 2000). Since these changes occurred in
preclinical stages of Alzheimer’s disease, inappropriate
activation of rab5a may be proposed as an important factor in
the pathogenesis of this disease. The enrichment of proteases
within early endosomes could conceivably facilitate the
proteolytic processing of amyloid precursor protein, and the
secretion of amyloid fragments, perhaps by a form of
recycling, could be especially rapid from this peripheral
compartment.
We are grateful to E. Millman for assistance with the Percoll
gradients. This work was supported by NIH grants R01HL50047
(B.J.K.) and K08HL03463 (R.H.M.). J.L.R. was supported by NIH
grants F32HL10150 and T32HL07676.
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