1 Department of Molecular Medicine, National Public Health Institute (KTL),
Biomedicum, PO Box 104, FIN-00251 Helsinki, Finland
2 Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310
Oslo, Norway
* Author for correspondence (e-mail: vesa.olkkonen{at}ktl.fi )
Accepted 4 December 2001
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Summary |
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Key words: Membrane trafficking, Endosome, Golgi apparatus, GTPase, Membrane fusion, Rab effector
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Introduction |
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A strikingly high number of Rab GTPases have been localized to endosomal
membrane compartments (reviewed by Rodman
and Wandinger-Ness, 2000). This most probably reflects the complex
organization and multiple sorting functions of endosomes
(Gruenberg and Maxfield, 1995
;
Mellman, 1996
). Material
internalized from the cell surface first enters the early endosome (EE) (also
called sorting endosomes), which are responsible for the dissociation and
sorting of receptors and ligands. Increasing evidence suggests that EEs also
receive cargo from the trans-Golgi network (TGN) through the
mannose-6-phosphate receptor (M6PR) pathway
(Press et al., 1998
;
Juuti-Uusitalo et al., 2000
).
These organelles are widely distributed in the cells and typically consist of
vacuole-shaped and tubular subdomains. Rab5, the most thoroughly characterized
member of the Rab GTPase subfamily, plays a key role in the transport of cargo
from the cell surface through clathrin-coated vesicles to the EE and in the
dynamic homotypic interactions between EE compartments
(Novick and Zerial, 1997
).
Receptors that are intended for reutilization are recycled to the cell surface
directly through the tubular EE subdomains or routed through a distinctive
population of tubular/vesicular compartments closely apposed to the
microtubule organizing center, denoted as perinuclear recycling endosomes
(Hopkins, 1983
;
Yamashiro et al., 1984
). The
recycling processes are regulated by Rab4 and Rab11, and Rab4 is suggested to
act at the level of the early `sorting' endosomes
(van der Sluijs et al., 1992
),
whereas Rab11 exerts a function in trafficking of cargo through the
perinuclear recycling endosomes (Ullrich
et al., 1996
; Ren et al.,
1998
). A recent study employing GFP-Rab fusion proteins and live
cell imaging depicted dynamic compartmentalization of different Rab proteins
within the same continuous membrane
(Sönnichsen
et al., 2000
): internalized transferrin was shown to move from
Rab5-positive to Rab5/Rab4 endosomes and then through Rab4/Rab11 containing
elements to the cell surface.
Molecules destined for degradation are sorted, most probably in the
vacuolar domains of EEs, to endocytic carrier vesicles that ferry them to the
late endosomes (LEs) (Gruenberg and
Maxfield, 1995). An alternative view is that EEs gradually mature
into LEs (van Deurs et al.,
1993
; van Deurs et al.,
1995
). LEs, which are characterized by a low lumenal pH and
extensive internal membranes rich in the unique phospholipid species
lyso-bis-phosphatidic acid (LBPA)
(Kobayashi et al., 1998
), form
another important sorting apparatus on the endocytic pathway: LEs receive
lysosomally destined cargo from the EEs and the trans-Golgi-network and
recycle receptors back to the TGN by a process regulated by Rab9
(Lombardi et al., 1993
). Rab7
localizes to LEs and data have been presented for its function in trafficking
from EEs to LEs (Feng et al.,
1995
; Mukhopadhyay et al.,
1997
; Press et al.,
1998
) and/or in the biogenesis of lysosomes
(Meresse et al., 1995
;
Bucci et al., 2000
).
A large number of Rab5 effector proteins have been identified
(Christoforidis et al., 1999).
One of these is the early endosomal antigen 1 (EEA1), a coiled-coil protein
whose other hallmark property is specific interaction with
phosphatidylinositol-3-phosphate (PtdIns-3-P)
(Simonsen et al., 1998a
;
Gaullier et al., 1998
). EEA1
plays a central role in the tethering of early endosomal elements
(Christoforidis et al., 1999
),
and it has recently been shown to directly interact with SNARE proteins
belonging to the syntaxin family (McBride
et al., 1999
; Simonsen et al.,
1999
). These findings have on one hand shed light on the
mechanisms by which specific membrane lipids modulate vesicular transport (see
Corvera et al., 1999
;
Huijbregts et al., 2000
) and
on the other provided clues to the mechanistic linkage between the Rab-based
membrane tethering machinery and the SNARE-based fusion apparatus.
Rab22 is a small GTPase whose two isoforms are expressed ubiquitously at
the tissue level and display the highest sequence similarity with Rab5
(Olkkonen et al., 1993;
Chen et al., 1996
). In the
present study we report the localization of Rab22a and its mutant forms that
are affected in the GTPase cycle, as well as the effects of their
overexpression on endosomal and Golgi morphology and transport processes in
the endocytic pathway. Furthermore, we show that, like Rab5, Rab22a interacts
with EEA1.
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Materials and Methods |
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Antibodies and other reagents
Rab22a was produced as an N-terminally His6-tagged fusion
protein from the pET11d expression plasmid (Invitrogen) in Escherichia
coli BL21(DE3) (Promega), purified on Ni-NTA-agarose (Qiagen) according
to a standard procedure and used for subcutaneous immunization of HsdRIVM:ELCO
rabbits. A His6-Glutathione S-transferase (GST)-tagged Rab22a
fusion protein was produced using the pGAT-4 expression vector (Johan
Peränen, Institute of Biotechnology, University
of Helsinki). The fusion protein was purified on glutathione-Sepharose
(Pharmacia), attached covalently to cyanogen-bromide-activated Sepharose 4B
(Pharmacia) and used for affinity purification of Rab22a antibodies by a
standard protocol. The anti-aspartylglucosaminidase (AGA) rabbit polyclonal
antibody was a gift from Anu Jalanko (National Public Health Institute,
Helsinki, Finland); the anti-LBPA and anti-hamster LAMP-1 monoclonal
antibodies were kindly provided by Jean Gruenberg (University of Geneva,
Switzerland). The anti-early endosomal antigen 1 (EEA1) rabbit polyclonal
antibody was described in Simonsen et al.
(Simonsen et al., 1998a); in
some experiments a human autoantiserum recognizing EEA1, kindly provided by
Ban-Hock Toh (Monash Medical School) was used. The anti-c-myc mAb was
produced as ascitic fluid in Balb/c mice from the hybridoma 9E10 purchased
from the European Collection of Animal Cell Cultures, and the monoclonal
anti-GM130 antibody was from Transduction Laboratories. Cycloheximide and
Wortmannin were purchased from Sigma and rhodamine-EGF from Molecular Probes.
Human holotransferrin (Sigma) was labelled with Alexa488 (Molecular
Probes) according to the manufacturer's instructions.
cDNA constructs and transfection
The wild-type (wt) Rab22a cDNA (GenBank accession no: Z22820) in pGEM-myc
(Olkkonen et al., 1993) was
used as template for site-directed mutagenesis using the Quickchange kit
(Stratagene) according to the manufacturer's instructions. The wt as well as
the S19N and Q64L mutant cDNAs carrying an N-terminal myc tag were tailored by
PCR to carry BamHI restriction sites flanking the open reading frame
at both ends. The cDNAs were inserted into the BamHI site of the CMV
promoter expression plasmid cDNA3.1 (Invitrogen), under the T7 promoter of
pGEM1 (Promega), or in pSFV1
(Liljeström
and Garoff, 1991
). Transfection of BHK-21 or COS-1 cells with the
pcDNA3.1 constructs was carried out with the Fugene 6 reagent (Roche)
according to the manufacturer's instructions using a transfection time of 24
hours. Alternatively, the proteins were expressed using recombinant Semliki
Forest viruses (SFV) prepared according to Olkkonen et al.
(Olkkonen et al., 1994
). In
some experiments, BHK-21, HeLa or Hep2 cells were infected with modified
Ankara T7 polymerase recombinant Vaccinia virus
(Sutter et al., 1995
) for 1
hour and then transfected for 6 hours with constructs in pGEM1, before
fixation and preparation for immunofluorescence microscopy. For protein
expression in E. coli untagged wt Rab22a cDNA with flanking
BamHI sites created by PCR was inserted into the BamHI site
of pGAT-4 or pGEX1
T (Pharmacia). pET-His-Rab22a was obtained by
cloning the NdeI-EcoRI and EcoRI-BglII
fragments from pGEM-myc-Rab22a into the NdeI and BamHI sites
of pET11d (Invitrogen). cDNA encoding the 209 N-terminal amino acids of EEA1
(1-209) was cloned into pMAL-c2 (New England Biolabs), for generation of
maltose binding protein (MBP)-EEA11-209 fusion protein
(Simonsen et al., 1998a
).
Immunofluorescence microscopy
The cells were fixed for 20 minutes at room temperature with 4%
paraformaldehyde, 250 mM Hepes, pH 7.4 and permeabilized for 20 minutes with
0.1% Triton X-100 in PBS. The primary antibodies diluted in 5% foetal calf
serum/PBS were incubated for 1 hour at 37°C and the bound antibodies were
detected with fluorescein-isothiocyanate (FITC)- or
tetramethylrhodamine-isothiocyanate (TRITC)-conjugated goat anti-rabbit or
anti-mouse F(ab)2 (Immunotech) or Cy-5-conjugated donkey
anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories). In some
experiments TRITC-conjugated donkey anti-mouse IgG, FITC-conjugated donkey
anti-mouse IgG or FITC- or TRITC-conjugated anti-human IgG (Jackson
ImmunoResearch Laboratories) were used. The specimens were mounted in Mowiol
(Calbiochem), 50 µg/ml 1,4 diazabicyclo-[2.2.2]octane (Sigma) and
investigated using a laser scanning confocal microscope (Leica SP1 or TCS NT).
In some experiments cells were permeabilized with 0.05% saponin prior to
fixation (Simonsen et al.,
1998b).
Yeast two-hybrid analysis
The yeast reporter strain L40 (Vojtek
et al., 1993) was cotransformed with the indicated pLexA and pGAD
plasmids, and ß-galactosidase activities of duplicate transformants were
determined as described in Guarente
(Guarente, 1983
).
In vitro binding of Rab22 to EEA1
For in vitro binding of EEA1, GST-tagged Rab5a or Rab22a produced from
pGEX1T were bound to Glutathione Sepharose beads (Pharmacia) in 20 mM
Hepes, 100 mM potassium acetate, 0.5 mM MgCl2, 1 mM DDT, 2 mM EDTA,
pH 7.2. The bound proteins were loaded with 10 µM GDP or GTP
S for 1
hour at 37°C. MBP-EEA11-209-fusion protein (1 µM) and 100 mM
MgCl2 were added on ice in the presence 10 mg/ml albumin and
incubated with the beads for 1 hour at 4°C, followed by washes with 20 mM
Hepes, 100 mM KAc, 5 mM MgCl2, 1 mM DDT, pH 7.2. The bound proteins
were eluted with 10 mM reduced glutathione for 20 minutes at room temperature
and analysed by SDS-PAGE and western blotting with anti-EEA1 antibodies.
Analysis of transferrin endocytosis
HeLa cells (used one day after seeding 5x104 cells per
coverslip) were transfected with pGEM-mycRab22a wt, -mycRab22a Q64L or
-mycRab22a S19N using the Vaccinia system. Six hours after the transfection,
the cells were washed three times in PBS and then incubated with
Alexa488-conjugated transferrin in Hepes-buffered medium containing
2 mg/ml BSA for 30 minutes at 37°C. The cells were then washed three times
in PBS and permabilized with 0.05% saponin prior to fixation in 3% PFA.
Analysis of EGF uptake and degradation
Hep2 cells (used one day after seeding 3x104 cells per
coverslip) were transfected with pGEM-mycRab22a wt, -mycRab22a Q64L, or
-mycRab22a S19N using the Vaccinia system. 5 hours after the transfection the
cells were incubated with rhodamine-labelled EGF (200 ng/ml) in Hepes-buffered
medium containing 2 mg/ml BSA for 1 hour at 37°C. The cells were then
washed three times with Hepes-buffered medium and then incubated further for 3
hours in Hepes-buffered medium containing 10 µg/ml cycloheximide. The cells
were thereafter washed three times in PBS and permabilized with 0.05% saponin
prior to fixation in 3% PFA.
TRITC-dextran uptake
To analyze the endocytic trafficking of TRITC-dextran, BHK-21 cells were
transfected with mycRab22a wt, S19N or Q64L cDNA in pcDNA3.1 for 24 hours. The
cells were incubated for 30 minutes on ice in air-medium [EAGLE MEM (Sigma),
0.292 g/l L-glutamine, 1 g/l glucose, 0.35 g/l NaHCO3, 10 mM Hepes,
pH 7.4], 5% foetal bovine serum, to prevent adsorption of the dextran
conjugate to the cell surface. Thereafter they were labeled for 1 hour at
37°C with 5 mg/ml TRITC-dextran (10 kDa; Molecular Probes) in air-medium
and fixed immediately or after 3 hours chase with complete medium and double
immunostained with anti-EEA1 and anti-LAMP-1 antibodies. Double stainings were
also carried out using the anti-myc mAb 9E10 for Rab22a and the above
compartment marker antibodies.
Analysis of the intracellular trafficking of AGA
BHK-21 cells were double transfected with mycRab22a wt or mutant pcDNA3.1
constructs and a human AGA/pSVPoly expression plasmid (Anu Jalanko, National
Public Health Institute, Helsinki, Finland) using Fugene 6 (Roche). After 24
hours, fresh culture medium containing 25 µg/ml cycloheximide was added and
the incubation was continued for 3 hours. Thereafter the cells were fixed and
triple stained with rabbit antibodies against AGA, human anti-EEA1
autoantiserum, and an anti-LBPA monoclonal antibody. In some triple stainings
the anti-LBPA mAb was replaced with the anti-myc mAb 9E10 to visualize the
myc-tagged Rab22a.
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Results |
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Interaction of Rab22a with EEA1
Prompted by the fact that Rab5 is the closest homologue of Rab22a (52%
identical) (Olkkonen et al.,
1993), we tested using the yeast two-hybrid system whether Rab22a
is also capable of interacting with EEA1
(Table 1). In the
ß-galactosidase reporter assay, we used Rab22a cDNAs from which the
C-terminal double cysteine motif was removed (marked with
C in
Table 1) to facilitate the
transport of the fusion proteins to the yeast nucleus and thus improve the
signal. This motif is subject to isoprenyl modification and is required for
the association of Rabs with membranes (see
Olkkonen and Stenmark, 1997
).
Rab22a Q64L
C displayed a strong interaction with full-length EEA1 and
even stronger with the N-terminal fragment (aa 1-209), whereas no interaction
was detected with a C-terminal fragment (aa 1257-1411). A similar result,
albeit with lower signals, was obtained when the EEA1 N- or C-terminal
fragments were used as baits and Rab22a Q64L
C as prey (not shown). The
Rab22a wt and S19N
C proteins showed no signal above background with any
of the EEA1 preys. The drastic difference between the Rab22a Q64L and S19N
mutants argued for the specificity of the observed interaction and suggested
that EEA1 only interacts with the GTP-bound form of Rab22a. Why Rab22 wt
C showed no significant interaction with EEA1 is not clear; one
possible explanation is that in yeast there could be a GAP activity that
stimulates GTP hydrolysis by Rab22a with high efficiency. It is noteworthy
that while Rab22a only seems to interact with the N-terminal region of EEA1,
Rab5a has been shown to have two binding sites, at both the N- and the
C-terminus of EEA1 (Simonsen et al.,
1998a
).
|
To verify the interaction of Rab22a and EEA1 biochemically, Rab5a and
Rab22a were expressed as fusion proteins with glutathione-S-transferase (GST)
in E. coli and bound on glutathione-sepharose beads. The fusion
proteins were then loaded with either GDP or the non-hydrolyzable analogue
GTPS, and pull-down experiments of soluble N-terminal fragment (aa
1-209) of EEA1 fused with maltose binding protein (MBP) were carried out
(Fig. 2). In this assay, the
GTP
S-loaded Rab5a was capable of specifically interacting with EEA1,
whereas the GDP-bound protein showed only very low affinity for the EEA1
fragment. Similarly, the GTP
S-bound Rab22a displayed specific binding
to EEA1; only a weak signal was detectable with the GDP-bound form of the
protein. On the basis of Ponceau red staining of the nitrocellulose filters,
approximately 5-10-fold less of the GTP
S-bound Rab22a than Rab5a was
required to obtain a given signal intensity in this binding assay.
|
To obtain more evidence for the interaction between Rab22a and EEA1 in live
cells, we employed the PtdIns-3-kinase inhibitor Wortmannin, which by
abolishing the generation of PtdIns-3-P detaches EEA1 from early endosomal
membranes. By overexpressing Rab5a it is possible to overcome the effect of
Wortmannin and lock EEA1 on the endosomal membranes
(Simonsen et al., 1998a).
After 30 minutes of treatment of BHK-21 cells with 100 nm Wortmannin, the
characteristic early endosomal EEA1 immunofluorescence staining was very weak
in mock-transfected BHK-21 cells, indicating detachment of EEA1 from the early
endosomal membranes (see Simonsen et al.,
1998a
). However, in cells overexpressing wt Rab22a, EEA1 remained
attached to the large vacuolar endosomes harbouring the overexpressed GTPase
(Fig. 3), arguing that Rab22a
indeed interacts with EEA1 and is capable of recruiting it onto the endosomal
membranes.
|
Effects of Rab22a overexpression on endocytic functions
The overexpression of Rab5a or its GTPase-deficient mutant form Q79L
(Hoffenberg et al., 1995) has
been shown to enhance the rate of endocytosis of transferrin (Tfn)
(Bucci et al., 1992
;
Stenmark et al., 1994
), and in
the case of Rab5a Q79L simultaneous inhibition of recycling to the cell
surface was observed (Stenmark et al.,
1994
). We therefore investigated the effects of Rab22a wt and the
S19N and Q64L mutants on the uptake and cellular distribution of Alexalabeled
human Tfn in HeLa cells. This cell line was chosen because of its abundant
expression of Tfn receptors and resulting efficient uptake of the human Tfn
conjugate. After Vaccinia T7 infection, the cells were transfected with
mycRab22a/pGEM1 constructs for 6 hours. Alexa-Tfn was then bound on the cells
and internalized for 30 minutes, and the labeled Tfn, as well as the expressed
GTPase, were visualized by confocal microcopy
(Fig. 4). In cells expressing
wt Rab22a, no change in the amount or distribution of internalized Alexa-Tfn
was observed as compared to untransfected cells
(Fig. 4A-E). Enlarged vacuolar
endosomes such as those induced in BHK cells were rarely observed in HeLa
cells, which are known to have smaller and more tubular endosomes; In these
cells the expressed GTPase showed a finer punctate staining pattern, which
overlapped with EEA1 (not shown) and the internalized Tfn
(Fig. 4C-E), as well as a
prominent perinuclear localization (Fig.
4C,F,I). Also in cells overexpressing Rab22a Q64L, the size of
Alexa-Tfn-positive endosomes appeared similar to that in untransfected cells.
However, the distribution of the marker was significantly altered: instead of
a relatively even cytoplasmic distribution, the Tfn-positive endosomal
structures were in Q64L expressing cells clustered near the cell surface at
the leading edges (Fig. 4F-H).
Expression of the S19N mutant had no obvious effect on the amount or
distribution of the internalized Tfn (Fig.
4I-K). The kinetics of transferrin internalization and recycling
was studied in COS-1 cells transfected with the Rab22a wt, Q64L or S19N
pcDNA3.1 constructs, using biotinylated human Tfn. As compared to
untransfected or mock-transfected (with the pcDNA3.1 vector) COS-1 cells, the
cells expressing Rab22a wt, S19N, or Q64L showed no significant difference in
the kinetics of either biotin-Tfn uptake or recycling (data not shown). These
results suggest that Rab22a does not regulate the transferrin cycle. However,
the redistribution of Tfn-positive structures to the leading edges of cells
that express the Q64L mutant indicates a possible role of Rab22a in regulation
of endosome motility.
|
The impact of Rab22a on membrane trafficking along the endocytic pathway was further investigated by monitoring the uptake and degradation of rhodamine-labeled epidermal growth factor (EGF) in Hep2 cells expressing the wt GTPase or its mutant forms. Hep2 provided a cell system feasible for this analysis owing to their efficient EGF binding and uptake. The rho-EGF was internalized for 1 hour, followed by a 3 hour chase before fixation of the cells and confocal microscopic observation of the labeled marker and the expressed GTPase. After 1 hour, the rho-EGF was found in Rab22a-positive endosomal structures, whose volume and distribution were highly similar in untransfected cells and those expressing wt Rab22a (Fig. 5A-C). However, a significant difference between untransfected and Rab22a expressing cells was apparent after a 3 hour chase: the rho-EGF fluorescence was no longer observed in the untransfected cells due to degradation of the growth factor, whereas it remained detectable in the Rab22a positive cells and colocalized significantly with the expressed GTPase (Fig. 5D-F). The same effect, albeit less pronounced, was observed in cells expressing Rab22a Q64L (Fig. 5G-L). Interestingly, although both Rab22a wt and Q64L clearly inhibited the degradation of the rho-EGF, the Q64L mutant did not colocalize with the marker after the 1 hour internalization period or after the 3 hour chase. Degradation of rho-EGF proceeded in a normal fashion in a majority of the cells expressing the S19N mutant (Fig. 5M-R).
|
The effects of Rab22a and the two mutants on rho-EGF degradation were quantified by calculating the proportion of the transfected cells that displayed rho-EGF positive endosomal elements after the 3 hour chase (Fig. 6). The highest frequency of such cells was observed in cultures expressing wt Rab22a (84%), the percentage in Q64L expressing ones being somewhat lower (70%). Also in cells expressing the S19N mutant the frequency (44%) was elevated compared to mock-transfected cells (17%), but the statistical variance of the results in the S19N expressing cells was high.
|
To elucidate the effects of Rab22a on fluid phase endocytosis, internalization and trafficking of TRITC-dextran were monitored in BHK-21 cells expressing wt Rab22a or its mutant forms. The transfected BHK-21 cells were allowed to endocytose TRITC-dextran for 1 hour, followed by a 3 hour chase incubation. The cells were then double immunostained for EEA1 and LAMP-1; in some experiments they were alternatively stained for Rab22a. In mock-transfected cells, dextran that had been internalized for 1 hour was found partly in EEA1-positive early endosomes and partly in LAMP-1-positive later compartments (not shown). During a 3 hour chase, it was chased to late structures, which showed no overlap with EEA1 but coincided to a large extent with LAMP-1 (Fig. 7A-E). In a majority of cells expressing wt Rab22a, the trafficking of TRITC-dextran proceeded as in the mock-transfected ones. Even though EEA1 was redistributed in the large vacuole-like endosomal structures induced by the GTPase, the internalized marker showed no colocalization with the EEA1 after the 3 hour chase but reached the LAMP-1-positive late endosomes/lysosomes (Fig. 7F-J). In cells expressing the Q64L mutant the situation was, however, different. The TRITC-dextran was found in the Rab22a Q64L-induced large vacuolar endosomes, which contained markers of both early (EEA1) and late (LAMP-1) endosomes (Fig. 7K-O), both after the 1 hour internalization and after the 3 hour chase. Mixing of the endosomal markers was most prominent in the largest Q64L-induced vacuolar structures. Expression of the S19N mutant had no apparent effect on transport of the internalized marker to LAMP-1-positive compartments (Fig. 7P-T). Quantification of the internalized TRITC-dextran after the 1 hour uptake period, carried out using the Leica LCS software, revealed no significant changes in fluid-phase uptake induced by Rab22a wt or either of the mutants as compared to untransfected cells (data not shown). These results suggest that the uptake or the trafficking of a fluid phase marker to late endocytic compartments marker is not markedly affected by wt Rab22a. However, the Q64L mutant causes mixing of early and late endosomal elements and creates an abnormal fusion compartment in which the fluid-phase marker accumulates.
|
To elucidate the possible effects of Rab22a on protein trafficking from the
biosynthetic pathway to endosomes/lysosomes, we monitored the effects of the
overexpressed wt or mutant proteins on the transport of coexpressed human
aspartylglucosaminidase (AGA), a lysosomal hydrolase that is routed to
endosomes via the M6PR-mediated pathway
(Enomaa et al., 1995;
Tikkanen et al., 1997
). The
proteins were expressed in BHK-21 cells for 24 hours, followed by a 3 hour
incubation in the presence of cycloheximide to chase the AGA from the
biosynthetic pathway to lysosomes. The cells were then triple immunostained
for AGA, EEA1 and LBPA. In some experiments, the LBPA staining was omitted and
the expressed Rab22a was visualized using anti-myc mAb. In mock-transfected
cells the AGA was chased to EEA1-negative vesicles/sites that contained the
late endocytic pathway marker LBPA (Fig.
8A-E). As in the case of TRITC-dextran, the transport of AGA into
late endosomes/lysosomes proceeded in a normal fashion in a majority of cells
overexpressing wt Rab22a and displaying the enlarged EEA1-positive early
endosomes (Fig. 8F-J). In most
(>85%) of the cells expressing Rab22a Q64L, however, the AGA accumulated in
abnormal vacuolar endocytic compartments, which were positive for both EEA1
and LBPA (Fig. 8K-O). Accumulation of AGA in the large spherical endosomes was not inhibited by 5 mM
mannose-6-phosphate included in the growth medium (data not shown), suggesting
that AGA was transported into these structures mainly from the TGN rather than
via the cell surface. Expression of Rab22a S19N had no apparent effect on the
trafficking of AGA (Fig. 8P-T).
Intriguingly, Rab22a Q64L was not detected on all large vacuolar endosomes
that contained accumulated AGA or TRITC-dextran (not shown).
|
Effects of Rab22a overexpression on the Golgi apparatus
Prompted by the observed abnormal trafficking of AGA, a marker originating
from the biosynthetic pathway, in cells transfected with the Rab22a Q64L
construct, we carried out triple staining of Rab22 wt or mutant-expressing
BHK-21 and HeLa cells for the expressed GTPase, for markers of the Golgi
apparatus (antibodies against GM-130 or mannosidase II) and for EEA1. While
Rab22a S19N was without effect on Golgi morphology, high-level expression of
wild type or Q64L Rab22a in HeLa cells caused a complete vesiculation of the
Golgi apparatus (Fig. 9). The
expressed wt and Q64L Rab22a were found to colocalize significantly with the
GM130-positive Golgi fragments. The dispersed Golgi elements did not contain
EEA1, indicating that no significant mixing had occurred between the Golgi and
endosomes. Fragmentation of the Golgi was also detected in BHK cells
expressing wt Rab22a or the Q64L mutant, but the effect was evident here only
in occasional cells (data not shown). The effect on Golgi morphology was
specific for Rab22, as it was not observed upon overexpression of wild-type or
GTPase-deficient mutants of Rab4, Rab5, Rab7 or Rab11 (not shown). These
results are consistent with the idea that Rab22a may regulate the trafficking
between the Golgi and early endosomes.
|
![]() |
Discussion |
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Interestingly, we found that, like Rab5, GTP-bound Rab22a specifically
interacts with the early endosomal FYVE-finger protein EEA1. However, although
Rab5 binds to both the N-terminal and the C-terminal regions of EEA1
(Simonsen et al., 1998a),
Rab22a only interacts with the EEA1 N-terminus. Furthermore, on the basis of
the results of our in vitro binding assay it seems that Rab22a shows higher
affinity for the EEA1 N-terminal region than Rab5 does. The finding that
Rab22a only binds to one end of EEA1 implies that its function is essentially
distinct from that of Rab5. The Rab5-EEA1 interaction has been shown to play a
central role in homotypic and heterotypic fusion in the early endocytic
pathway (Christoforidis et al.,
1999
), a process in which Rab5 resides on both apposed membranes
and EEA1 can be linked to Rab5 at its both ends, leading to formation of
symmetrical bridges of Rab5 and the bound effector. The Rab22a-EEA1
interaction alone cannot lead to the generation of such symmetrical bridges.
Instead, Rab22a could participate in membrane interactions in which the other
end (C-terminus) of EEA1 is bound to PtdIns-3-P and Rab5. Accordingly, Rab22
should, in principle, have the capacity to control fusion of early endosomes,
as suggested by our morphological data. However, if active Rab22a is also
localized on transport vesicles orginating, for example, from the trans-Golgi
network, the interaction with the N-terminus of EEA1 may provide a mechanism
through which these vesicles could be tethered at their early endosomal
targets.
In studies employing Alexa- or biotin-conjugated transferrin to monitor the
receptor-mediated uptake or recycling of this protein, transient
overexpression of wt Rab22a or the Q64L or S19N mutants had no significant
effect. Therefore, it seems that the function of early endosomal compartments
in receiving cargo from the cell surface or recycling molecules is not
severely disturbed by overexpression of the GTPase. Further, the lack of
detectable effects on transferrin recycling is in accordance with the observed
lack of colocalization between Rab22a and the recycling endosome marker Rab11.
However, overexpression of the GTPase-deficient Rab22a Q64L did have a marked
effect on the intracellular distribution of Alexa-Tfn: instead of being
dispersed in the cytoplasm as in non-transfected cells, the
Alexa-Tfn-containing endosomal elements were found clustered at the leading
edges of the cells. This indicates that Rab22a may, like its closest homologue
Rab5 (Nielsen et al., 1999),
play a role in regulating the motility and intracellular distribution of early
endosomal elements. Intriguingly, the expressed Rab22a Q64L did not colocalize
with the peripherally localized Alexa-Tfn-positive elements. This could be due
to 1) the ability of the GTPase-deficient mutant to cause a change in the
motility of the Tfn-containing endosomes through a very transient interaction
with these elements or, more likely, to the 2) sequestration by Rab22a Q64L of
a cytosolic factor that is required for the normal localization of the
Tfn-containing endosomes.
As in the experiments measuring Tfn uptake, no obvious effect of wt or
mutant Rab22a was observed on the internalization of rhodamine-EGF from the
surface of Hep2 cells. However, especially the wt Rab22a but also the Q64L
mutant were found to inhibit rho-EGF degradation. It has been reported that
EGF-EGF receptor complexes are routed to lysosomal degradative compartments
through distinct maturing multivesicular body (MVB)-type, LAMP- and
M6PR-negative, structures (Futter et al.,
1996). The results thus suggest that wt Rab22a and the activated
Q64L mutant interfere with the formation of such MVBs or their ability to
interact with lysosomes. Alternatively, changes in the endocytic pathway
organization induced by Rab22a (see below) may have disturbed the degradative
function of lysosomes.
In BHK-cells, the Q64L mutant disturbed the transport of AGA, a lysosomal
hydrolase routed to endosomes via the M6PR pathway, and an internalized
fluid-phase marker, TRITC-dextran, which accumulated in large
vacuole-appearing structures. These structures contained markers of both early
and late endosomes. Formation of such abnormal fusion compartments is not
unique to Rab22a: it has also been detected in cells expressing the
GTPase-deficient Q79L mutant of Rab5a
(d'Arrigo et al., 1997) or an
ATPase-deficient mutant of mouse SKD1, a homologue of S. cerevisiae
Vps4p (Yoshimori et al.,
2000
). The data indicate that upon increasing overexpression of
Rab22a Q64L, the enlarged early endosomes appear first and then also start to
acquire late endosomal markers. It is thus possible that the process is due to
enhanced retrograde transport from late to early compartments. If one, on the
other hand, considers the models of endosomal maturation and the possibility
that maturing early endosome subdomains could acquire late endosomal
constituents by kiss-and-run type interactions
(Storrie and Desjardins,
1996
), the Rab22a Q64L mutant phenotype could arise from
abnormally enhanced kiss-and-run contacts.
Strikingly, the wt protein and the Q64L mutant also caused, in HeLa cells,
a complete vacuolization of the Golgi apparatus and a similar but less
prominent effect in BHK cells. This unique finding, together with the partial
colocalization of Rab22a with a Golgi marker in HeLa cells, suggests that
Rab22a may have a function in the communication between Golgi and early
endosomes (see Press et al.,
1998; Juuti-Uusitalo et al.,
2000
; Johannes and Goud,
2000
; Sandvig and van Deurs,
2000
). The finding that EEA1, a binding partner of Rab22a, also
binds syntaxin 6 (Simonsen et al.,
1999
), provides a possible mechanistic link between Rab22 function
and TGN to endosome trafficking. Syntaxin 6 localizes mainly to the
trans-Golgi network (Bock et al.,
1997
) and is implicated in several transport events involving the
TGN (Wendler and Tooze, 2001
).
It is interesting to note that a possible yeast homologue of EEA1, Vac1p,
regulates TGN to endosome trafficking
(Peterson et al., 1999
;
Tall et al., 1999
). Thus,
Rab22a-induced recruitment of EEA1 as a tethering factor in heterotypic
contacts between TGN-derived and early endosome membranes could possibly
facilitate the local assembly of a syntaxin 6-containing SNARE complexes.
Further, it will be interesting to elucidate if Rab22a also binds rabenosyn-5
(Nielsen et al., 2000
), a
novel effector of Rab5 and a functional homologue of yeast Vac1p. In order to
resolve the directionality of the trafficking step(s) regulated by Rab22a, it
will be informative to study the role of this protein in the intoxication by
Shiga toxin, which is believed to enter the TGN via early endosomes
(Johannes and Goud, 2000
;
Sandvig and van Deurs,
2000
).
The question of why Rab22a causes a fragmentation of the Golgi apparatus is
intriguing. On the basis of studies of Rab1, it has been suggested that the
same Rab could bind tethering factors at both the donor and the acceptor
compartments in a vesicular transport step
(Moyer et al., 2001). Thus,
Rab22a might interact with an unidentified Golgi tethering protein, in
addition to with EEA1 (see Barr,
1999
), and when present in excess amounts, it disturbs the
function of this factor, resulting in organelle fragmentation. On the other
hand, the observed effect on endosome motility indicates that Rab22a may
interfere with the function of microtubule motor proteins or proteins
tethering membrane organelles to microtubules, which could also lead to
redistribution of the Golgi apparatus.
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Acknowledgments |
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References |
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