Experimental Cell Research 304 (2005) 339 – 353
www.elsevier.com/locate/yexcr
Overexpression of Rab22a hampers the transport between
endosomes and the Golgi apparatus
Rosana Mesaa, Javier Magadána, Alejandro Barbierib,1, Cecilia Lópeza,
Philip D. Stahlb, Luis S. Mayorgaa,*
a
Laboratorio de Biologı́a Celular y Molecular, Instituto de Histologı́a y Embriologı́a (IHEM-CONICET),
Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina
b
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Received 16 September 2004, revised version received 9 November 2004
Available online 16 December 2004
Abstract
The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set
of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and
inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic
transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP
(Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine–dextran to lysosomes,
the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the
endosome–lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera
toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in
endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting
fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network
(TGN).
D 2004 Elsevier Inc. All rights reserved.
Keywords: Intracellular transport; Endocytosis; Rab proteins; Golgi apparatus; Cholera toxin
Introduction
Early endosomes form a highly dynamic tubulovesicular
compartment that actively receives material from the plasma
membrane and sorts it to several destinations (for a review,
see Ref. [1]). Most soluble material, together with some
membrane lipids and proteins, are directed to lysosomes for
digestion. Many receptors and membrane proteins are
recycled back to the plasma membrane, either directly or
* Corresponding author. Instituto de Histologı́a y Embriologı́a (IHEM),
Casilla de Correo 56, (5500) Mendoza, Argentina. Fax: +54 261 4494117.
E-mail address: [email protected] (L.S. Mayorga).
1
Present address: Department of Biological Sciences, Florida
International University, University Park, Miami, FL 33199, USA.
0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2004.11.017
through the endocytic recycling center. Finally, other sets of
membrane lipids and proteins are directed to the Golgi
apparatus. This complex sorting process is only partially
understood. However, it is clear that Rab proteins are key
protagonists for the correct trafficking along the endocytic
pathway [1–3].
Rab function is regulated by cycles of GTP binding and
hydrolysis that result in conformational changes in the
protein that promote specific contacts with different
effectors [4]. Transport vesicles carry Rab proteins with
bound GTP. After membrane fusion, GTP hydrolysis
stimulated by specific GAPs (GTPase-activating proteins)
generates GDP-bound Rabs. A cytosolic protein called GDI
(GDP dissociation inhibitor) extracts Rabs from the
membranes and delivers them to the compartments of origin
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R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
where they are subsequently reactivated by Rab-specific,
nucleotide exchange factors.
Several Rabs are present in the endocytic pathway where
they are thought to regulate different steps [1]. Rab5, the
best-characterized Rab in endocytosis, associates with early
endosomes, is necessary at early steps in the endocytic
process, and is required for homotypic fusion among early
endosomes [5]. Direct recycling of receptors from these
vesicles to the plasma membrane requires Rab4 [6], while
recycling via the perinuclear recycling center depends on
Rab11 [7]. Transport from early endosomes to lysosomes is
directed by Rab7 [8]. More that one pathway functions in
transport from endosomes to the Golgi apparatus. The bestcharacterized Rab functioning in this route is Rab9, which is
required for the transport of the cation independent mannose
6-phosphate receptor (CI-M6PR) from late endosomes to
the Golgi apparatus [9]. The other path is from early and
recycling endosomes to the trans-Golgi network (TGN) and
is regulated by Rab6a’ [10].
Rab22a was first described as associated with the
endocytic pathway 11 years ago [11]. Recently, a more
detailed characterization has been carried out [12–14].
However, at present, very little is known about Rab22a
function and conflicting observations have been reported in
different cell types. It is clear from these publications that
Rab22a has a role in the early endocytic pathway [12,14]
and in the recycling of some membrane proteins, such us the
major histocompatibility complex class I (MHCI) to the cell
surface [13]. Colocalization studies show that the protein is
present in early endosomes and that physically interacts
with EEA1, one of the best-characterized effector of Rab5
[14]. However, Rab22a also colocalizes with the CI-M6PR
[12], a protein that is present mainly in the TGN and in late
endosomes in CHO cells. The aim of the present work was
to characterize the function of Rab22a by analyzing the
intracellular transport along the endocytic pathway in CHO
cells overexpressing the wild-type protein and two mutants.
Our results show that overexpression of Rab22a wild-type
hampers trafficking from endosomes to the Golgi apparatus
inducing the accumulation of CI-M6PR and cholera toxin in
early endocytic compartments. The positive mutant causes
the same effect and, in addition, is mislocated to late
endocytic compartments, including lysosomes.
Materials and methods
USA). Rabbit anti-CI-M6PR antibody was generously
provided by Stuart Kornfeld (St. Louis, MO, USA). Rabbit
anti-cathepsin D antibody was a generous gift from William
Brown (Utica, NY, USA). Mouse anti-syntaxin 6 and antiEEA1 were from Transduction Laboratories (Lexington,
KY, USA). Secondary anti-mouse and anti-rabbit antibodies
labeled with Alexa Fluor 546 or Alexa Fluor 350 were
purchased from Molecular Probes. All other reagents were
from Sigma (St. Louis, MO, USA).
Plasmids
The cDNA for Rab22a was kindly provided by Marino
Zerial (Heidelberg, Germany). Rab22a cDNA was amplified by PCR and subcloned as BamHI fragments into the
pGEX-2t plasmid (Amersham Pharmacia Biotech, Uppsala,
Sweden). Rab22a mutants (Q64L and S19N) were obtained
by site directed mutagenesis (QuickChange kit from
Stratagene, La Jolla, CA, USA). Mutations were verified
by sequencing the inserts. Wild type and mutants were
subcloned in the BamHI site of pEGFP-C1 and pECFP-C1
(Clontech, Palo Alto, CA, USA). Rab5a was subcloned in
the HindIII and BamHI sites of pEYFP-C1. pEGFP-rab7
was obtained from Bo van Deurs (Copenhagen, Denmark)
and subcloned in the HindIII and XbaI sites of pYFP-C1.
The plasmid pEYFP-GalNAc-T1–27 was a generously gift
from Hugo Maccioni (Córdoba, Argentina). The plasmid
pEYFP-rab9 was kindly provided by Suzanne Pfeffer
(Standford, CA, USA).
Cell culture, transfection, and treatments
CHO cells were grown in a-minimal essential medium
(aMEM) supplemented with 10% fetal bovine serum. Before
transfection, cells were plated on coverslips for 24 h. Cells
were washed three times with serum-free aMEM and
transfected or cotransfected with 1 Ag/ml of each purified
plasmidic DNA using the LipofectAmine reagent (GIBCO
BRL, Gaithersburg, MD, USA) according to the manufacturer’s instructions. To generate stable cell lines expressing
Rab22a WT.GFP, Rab22a S19N.GFP, or Rab22a Q64L.GFP,
cell clones resistant to 1 mg/ml G418 antibiotic (GIBCO
BRL) were selected. CHO cell clones expressing Gal-T2-HA
(UDP-Gal:GA2/GM2/GD2 galactosyltransferase) and GalNAc-T-myc (UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltranferase) were generously provided by José
Luis Daniotti (Cordoba, Argentina) [15].
Reagents
Labeling of subcellular compartments
Lysine-fixable dextran cascade blue (B-dextran), lysinefixable dextran tetramethylrhodamine (Rh–dextran), Alexa
594-conjugated cholera toxin subunit B (CTxB), rhodamine-labeled epidermal growth factor (Rh-EGF), and
LysoTracker Blue DND-22 were from Molecular Probes
(Eugene, OR, USA). Lipoprotein-deficient fetal calf serum
(LPDS) was from Cocalico Biologicals (Reamstown, PA,
To label early compartments loaded with the fluid phase
marker Rh–dextran, CHO cell monolayers were washed
three times with uptake medium (Basal Medium Eagle,
20 mM HEPES, pH 7.4, 5 mg/ml BSA) and incubated
5 min at 378C in uptake medium containing 1 mg/ml Rh–
dextran. To label lysosomal compartments, CHO cell
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
monolayers were washed three times with uptake medium
and incubated 1 h at 378C with uptake medium containing
1 mg/ml Rh–dextran or B-dextran followed by 3 h chase in
the absence of dextran. Lysosomes were also labeled by
incubating the cells for 20 s at 378C with 1 AM LysoTracker
Blue DND-22 just before fixation. To immunostain compartments containing CI-M6PR, cathepsin D, syntaxin 6,
GalNAc-T-myc, or EEA1, cells were fixed in 4% paraformaldehyde dissolved in PBS containing 1 mM MgCl2,
0.2 mM CaCl2 (PBS-CM) for 20 min at room temperature,
and permeabilized with 0.5% Triton X-100 in PBS-CM.
Cells were washed once with PBS-CM and incubated for
15 min at room temperature in the same buffer containing
50 mM NH4Cl. Primary antibodies were diluted 1:100 in
PBS-CM containing 1% BSA and incubated with the cells
overnight at 48C. Secondary antibodies were diluted 1:300
in PBS-CM containing 1% BSA and incubated with cells
for 2 h at 208C. Washes between antibodies were carried out
with PBS-CM containing 1% BSA.
Kinetics of internalization of Rh–dextran
CHO cell monolayers plated on coverslips for 24 h were
washed three times with uptake medium. Lysosomes were
labeled as described above with B-dextran (1 h uptake, 3 h
chase). Rh–dextran (1 mg/ml) was then internalized for
5 min at 378C. Cells were washed three times with uptake
medium and incubated for 15, 30, 45, or 90 min. Finally, the
cells were washed five times with ice-cold PBS and fixed.
At least 250 vesicles containing Rh–dextran were counted in
at least 25 cells. These vesicles were classified according to
whether they contained Rab22a or B-dextran or both.
Results were expressed as a percentage of the total vesicles
counted containing Rh–dextran.
Fluorescence microscopy
Unfixed and fixed cells were observed in an Eclipse
TE300 Nikon microscope equipped with a Hamamatsu Orca
100 camera. Images were taken with three sets of filters
(excitation 510–560, barrier 590 for tetramethylrhodamine,
Alexa Fluor 546 and Alexa 594-conjugated CTxB; excitation 450–490, barrier 520 for GFP; and excitation 330–380,
barrier 420 for B-dextran, LysoTracker Blue DND-22 and
Alexa Fluor 350). Cells expressing constructs of CFP or
YFP were observed with an LSM-510 confocal microscope
(Carl Zeiss, Jena, Germany). Image processing and fluorescence quantification were carried out using the MetaMorph 4.5 Imaging System (Universal Imaging, West
Chester, PA). Final images were compiled with Adobe
Photoshop 5.0.
Endocytosis of cholera toxin
Endocytosis of cholera toxin was carried out essentially
as described by Holtta-Vuori et al. [16]. CHO cells
341
expressing Gal-T2-HA and GalNAc-T-myc were incubated
with 1 Ag/ml Alexa 594-conjugated cholera toxin (CTxB) in
uptake medium for 1 h on ice at 48C. After washing three
times with uptake medium, the cells were incubated in
serum-free culture medium supplemented with 0.01% BSA
for 10 min or 1 h at 378C and fixed. To immunostain the
distal Golgi, GalNAc-T-myc was labeled with an anti-myc
antibody followed by Alexa Fluor 350-conjugated secondary antibody. To quantify the intracellular cholera toxin not
associated with the Golgi apparatus, the fluorescence
emitted by CTxB that colocalized with GalNAc-T-myc
was subtracted and the result expressed as a percentage of
the total fluorescence present in the cell. At least 40
transfected and untransfected cells were quantified for each
coverslip.
LDL, BSA, and EGF hydrolysis assays
LDL was radioiodinated by the iodine monochloride
method [17]. CHO cells were grown in monolayer in 35mm dishes (80% confluence) containing a-MEM supplemented with 10% lipoprotein-deficient fetal calf serum
(LPDS). Cells were then incubated in uptake medium
containing 15 Ag/ml 125I LDL 2 h at 48C in the presence
or absence of 50-fold excess of unlabeled LDL. The cell
monolayers were washed five times with ice-cold 1% PBSBSA and five times with ice-cold PBS. Afterwards, the
wells were incubated 6 h at 378C in a-MEM supplemented
with 10% LPDS to allow sufficient 125I-labeled degradation
products to accumulate in the medium. The media were
collected and dead cells removed by low-speed centrifugation. The supernatants were harvested and trichloroaceticacid-soluble radioactivity in all samples was measured.
Digestion was calculated as a percentage of the total
radioactivity bound to the cells that were recorded as acid
soluble after the 6-h incubation. To compare different
experiments performed with different 125I LDL preparations, the data were normalized by dividing the values by
the average digestion measured for all the cells types within
each experiment (range 27–48%). Nonspecific binding in
cells was less than 5% of the total binding. In order to
follow BSA degradation, BSA was radiolabeled with 125I
using chloramine T [18]. CHO cells monolayers in 35-mm
dishes at 60–75% confluence were allowed to internalize
radioactive BSA for 1 h at 208C. The cells were then
washed five times with ice-cold PBS containing 1% BSA
and incubated for 30 min at 378C. At the end of the
incubation, the cells were solubilized in PBS containing 1%
Triton X-100. Trichloroacetic-acid-soluble radioactivity was
measured in all the samples. Digestion was calculated as the
percentage of the total radioactivity internalized by cells that
became acid soluble after the 30-min incubation. To
compare different experiments performed with different
125
I BSA preparations, the data were normalized by dividing
the values by the average digestion measured for all the cells
types within each experiment (range 20–29%).
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To assess the effect of Rab22a on EGF digestion, a
protocol similar to that described by Kauppi et al. [14] was
used. In brief, CHO cells expressing human EGF receptor
(kindly provided by Jeffrey E. Pessin, St. Louis, USA) were
transiently transfected with Rab22a WT.GFP, Rab22a
S19N.GFP, or Rab22a Q64L.GFP as described above. This
cell line did not tolerate the Q64L mutant; therefore, the
experiments were performed only with the WT- and S19Nexpressing cells. Twenty-four hours after transfection, the
cells were incubated for 10 min or 1 h at 378C in Dulbecco’s
modified eagle medium buffered with HEPES containing
1% BSA and 400 ng/ml of rhodamine-labeled EGF. After
several washes with HEPES-buffered medium, the cells
were incubated in aMEM supplemented with 10% fetal
bovine serum for 0 or 3 h, washed several times with the
same medium and with PBS and fixed. The amount of
rhodamine fluorescence per cell was quantified in at least 20
transfected and untransfected cells for each condition by
using the MetaMorph 4.5 Imaging System.
Results
Relationship between Rab22a and other endocytic Rabs
(Rab5, Rab7, and Rab9)
Previous results from our laboratory and others indicate
that Rab22a localizes to early endosomes and recycling
endosomes containing MHCI [11–14]. Less clear is the
function of this protein in intracellular trafficking. The
results obtained thus far largely depend on the cell line used
for the studies. Our goal was to perform a careful characterization of the effect of Rab22a overexpression in a single
cell line. CHO cells were selected because they feature a
well-characterized endocytic pathway. As a first step, the
relationship between Rab22a with other Rabs that are
characteristic of different compartments along the endocytic
pathway was assessed in this cell line. For this purpose,
Rab22a was subcloned in the pECFP-C1 plasmid and
coexpressed with Rab5, Rab7, and Rab9 tagged with YFP.
Fig. 1 shows that Rab22a presents a prominent colocalization with Rab5 (Figs. 1A–C) but a very limited one with
Rab7 (Figs. 1D–F). Interestingly, although colocalization
with Rab9 was rare, several Rab22a-positive structures were
observed in close proximity to Rab9-containing vesicles
(Figs. 1G–I and insets). These results indicate that Rab22a
associates with early endosomes but is excluded from late
endocytic compartments enriched in Rab7. Rab22a may be
present in some in Rab9-containing compartments.
Overexpression of Rab22a does not block transport to
lysosomes
Because Rab22a presents a prominent localization in
endosomes, it is reasonable to speculate that it might affect
one or more of the trafficking pathways associated with this
compartment. A series of experiments were carried out to
assess the effect of this GTPase in the transport to lysosomes
of internalized macromolecules. Lysosomes of CHO cells
were labeled with dextran cascade blue (B-dextran, 1 h
uptake, 3 h chase). Afterwards, the kinetics of arrival of a
5-min pulse of rhodamine-tagged dextran (Rh–dextran) to
the blue (i.e., lysosomal) compartments was followed in
CHO cells overexpressing GFP alone, Rab22a WT.GFP,
Rab22a Q64L.GFP (a mutant with reduced GTPase activity), and Rab22a S19N.GFP (a mutant with reduced affinity
for GTP).
Overexpression of wild-type Rab22a results in the
formation of large vesicles that were loaded with the
endocytic marker after the 5-min pulse but not with the
lysosomal marker (Figs. 2A–D). After 45-min chase, Rh–
dextran moved to the B-dextran-positive Rab22a-negative
compartments (Figs. 2E–H). A different picture was
observed in cells overexpressing the Rab22a Q64L mutant.
In these cells, Rh–dextran remained in Rab22a-positive
vesicles during the entire chase period (Figs. 2M–P). Unlike
the wild-type protein, the positive mutant was present in
vesicles loaded with B-dextran (the lysosomal marker).
Several explanations could account for these observations.
(i) The complete endocytic pathway may have fused into a
single compartment containing early and late markers. (ii)
Transport to lysosomes may be blocked in these cells.
Therefore, the lysosomal marker may have been trapped,
together with Rh–dextran, in endosomes. (iii) The positive
mutant may associate with both endosomes and lysosomes.
The time course analysis indicates that Rh–dextran was first
present in B-dextran-negative compartments (Figs. 2J–L),
and that after 45-min chase the two dextrans mixed (Figs.
2N–P). At early time points, the positive mutant clearly
labeled two different vesicle populations, one containing
Rh–dextran and another loaded with B-dextran (insets Figs.
2I–L). These observations indicate that Rh–dextran moved
along different compartments in these cells, a conclusion
that is compatible only with the third possibility.
A quantitative analysis of Rh–dextran transport to
lysosomes indicates that the same kinetics were observed
in cells expressing wild-type Rab22a, the negative mutant,
or GFP alone (Fig. 3A). In contrast, the kinetics of arrival to
lysosomes was slower in cells transfected with the positive
mutant (Fig 3A). In Fig. 3B, it is evident that Rh–dextran
left the early compartments labeled with Rab22a WT few
minutes after internalization. In contrast, Rh–dextran
remained inside Rab22a Q64L-positive vesicles even after
90 min of chase (Fig. 3B). However, it should be noted that
initially Rh–dextran was present in Rab22a Q64L-positive
vesicles lacking the lysosomal marker (B-dextran) and
moved with time to Rab22a Q64L-positive vesicles loaded
with B-dextran (Fig. 3C).
The same kinetic analysis was performed by labeling
lysosomes with LysoTracker blue, a fluorescent probe that
accumulates in acidic compartments. The results obtained
were similar to those presented in Fig. 2. Rh–dextran was
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
343
Fig. 1. Colocalization of Rab22a WT with other endosomal Rab proteins. CHO cells were cotransfected with pECFP-rab22a WT and pEYFP-rab5 (A–C),
pEYFG-rab7 (D–F), or pEYFP-rab9 (G–I). Twenty-four hours after transfection, the distribution of the fluorescent proteins was recorded in a confocal
microscope. Strong colocalization between Rab5 and Rab22a was observed (A–C). On the contrary, Rab7 and Rab22a were observed in separated
compartments (D–F). Colocalization with Rab9 was not prominent, but some Rab22a-positive structures were observed in close proximity to Rab9-containing
vesicles (G–I and insets). Scale bars = 7 Am.
found initially in LysoTracker-negative compartments and
with time it moved to LysoTracker-positive compartments
both in cells expressing Rab22a WT and Rab22a Q64L.
Also, the positive mutant was present in early and late
(LysoTracker-positive) compartments (data not shown).
These observations indicate that overexpression of Rab22a
WT and Rab22a S19N does not affect the kinetics of
transport to lysosomes. However, the positive mutant
delays—but does not block—transport to lysosomes.
Interestingly, the results show that although the positive
mutant is present in both endosomes and lysosomes, these
compartments do not mix.
Overexpression of Rab22a does not alter the lysosomal
function
According to our results, overexpression of Rab22a
does not block the transport to lysosomes of endocytosed
material. We wondered whether the trafficking of acid
hydrolases, which are delivered to endosomes from the
TGN en route to lysosomes, was affected by Rab22a.
Therefore, the localization of cathepsin D, a wellcharacterized lysosomal enzyme, was studied. The enzyme
was found in perinuclear vesicles—similar to what was
observed in untransfected cells—that did not colocalized
with Rab22a wild type (Figs. 4A–D). Triple colocalization
studies of Rab22a, B-dextran chased for 3 h, and
cathepsin D showed that the lysosomal distribution of
the enzyme was not altered in cells expressing the wildtype protein (Figs. 4A–D) or either of the mutants (Figs.
4E–L). As expected, the positive mutant caused an
enlargement of cathepsin D-containing vesicles and
extensively labeled these compartments (Figs. 4E–H).
We conclude that there is no gross defect in the transport
of cathepsin D to lysosomes in Rab22a-transfected CHO
cells.
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R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
Fig. 2. Rab22a WT and the positive mutant do not block the transport of internalized material to lysosomes. Stable CHO cell lines expressing Rab22a WT (A–
H) or Rab22a Q64L (I–P) were incubated for 1 h at 378C in the presence of 1 mg/ml of cascade blue dextran (uptake), washed and incubated 3 h at 378C
(chase) in order to label lysosomes. The cells were allowed to internalize rhodamine dextran for 5 min at 378C and fixed (5 min rows) or chased for 45 min (50
min rows) before fixation. The fluorescence of GFP-Rab22a (WT or Q64L column), rhodamine dextran (Rh–dextran column), and cascade blue dextran in
lysosomes (lysosomes column) was recorded in a Nikon inverted microscope. Superposition of the three fluorescence channels is shown in the overlap column.
Notice that in Rab22a WT-expressing cells (A–H), Rh–dextran after 5 min of internalization is present in Rab22a-containing compartments lacking the
lysosomal marker. After 45 min of chase, the Rh–dextran moved to lysosomal compartments lacking Rab22a WT. In cells expressing the positive mutant (I–P),
Rh–dextran was present at early times in Rab22a Q64L-positive vesicles lacking the lysosomal marker. After the 45-min chase, Rh–dextran moved to
lysosomal marker-containing compartments. The difference was that these vesicles were also decorated with Rab22a Q64L (M–P). Scale bars = 7 AM. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
We have shown that the transport of endocytosed macromolecules from the cell surface and of acid hydrolases to
lysosomes is not impaired in cells overexpressing Rab22a.
Therefore, the function of lysosomes should not be affected.
In a previous report [12], we have observed that digestion of
HRP is not significantly altered by overexpression of
Rab22aWT or the positive and negative mutants. To
strengthen the observation that the lysosomal function is
preserved, we examined the digestion of BSA (a protein
internalized mostly by fluid phase endocytosis in CHO cells,
which is highly sensitive to lysosomal proteolysis) and of
LDL (which enters cells by receptor-mediated endocytosis)
in CHO cells overexpressing Rab22a WT and their mutants.
As shown in Fig. 5, overexpression of Rab22a did not
significantly affect digestion of BSA or LDL. The results
indicate that despite the dramatic morphological alteration of
endocytic compartments observed in Rab22a-expressing
cells, lysosomal digestion is not significantly altered.
Other authors have reported that overexpression of
Rab22aWT and the Q64L mutant strongly increases the
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
345
percentage of cells labeled with Rh-EGF after an internalization of 1h followed by 3 h of chase [14]. This observation
has been interpreted as an inhibitory effect of Rab22a on EGF
digestion. To assess the uptake and digestion of this ligand in
CHO cells, a cell line expressing the human EGF receptor
was transiently transfected with Rab22a WT and Rab22a
S19N. We could not transfect with Rab22a Q64L because the
positive mutant was toxic for this cell line. Rh-EGF was
internalized for 10 min at 378C to label early compartments
or for 1 h followed by 3 h chase. Rh-EGF presented a strong
colocalization with Rab22a WT after a 10-min uptake (data
not shown). The localization was only partial after 1 h of
continuous uptake, suggesting that some EGF had left early
endosomes (data not shown). After the 3-h chase, Rh-EGF
fluorescence was barely observed in the cells, indicating that
the ligand was efficiently removed in untransfected and
Rab22a (WT and S19N)-transfected cells (Fig. 5).
Rab22a disturbs the transport from endosomes to the TGN
Fig. 3. Kinetics of transport of internalized fluid phase marker to lysosomes
in cells overexpressing Rab22a wild type and mutants. In stable CHO cell
lines expressing GFP alone or coupled to Rab22a WT, Rab22a Q64L, or
Rab22a S19N, the kinetics of arrival of a 5-min pulse of rhodamine–dextran
(Rh–dextran) to lysosomal compartments was recorded in a series of
experiments similar to the one shown in Fig. 2. For each chase time (0, 15,
30, 45, or 90 min), the total number of vesicles containing Rh–dextran were
counted in at least 25 cells. These vesicles were classified according to
whether they were decorated with Rab22a (wild type or Q64L; the S19N
mutant is mostly cytosolic) and whether they contained the lysosomal
marker (B-dextran internalized for 1 h and chased for 3 h). (A) The
percentage of Rh–dextran-positive vesicles that contained the lysosomal
marker is plotted as function of time in cells expressing GFP alone (empty
circles), Rab22a WT (solid circles), Rab22a S19N (empty squares), or
Rab22a Q64L (solid squares). Notice that only the positive mutant delayed,
although did not block, transport to lysosomes. (B) The percentage of Rh–
dextran-positive vesicles that were decorated with Rab22a WT (circles) or
Rab22a Q64N (squares) is plotted as a function of time. It is evident that the
fluid phase marker quickly leaves Rab22a WT-containing vesicles but
remains in Rab22a Q64L-positive vesicles. (C) The total percentage (solid
squares) of vesicles containing the fluid phase marker that were decorated
with Rab22a Q64L were classified according to whether they contained (full
triangles) or lack (empty triangles) B-dextran. It is clear that, with time, Rh–
dextran moved from vesicles lacking to vesicles containing the lysosomal
marker. This experiment was repeated three times with similar results.
The CI-M6PR shuttles between the TGN and endosomes
transporting lysosomal enzymes. We have previously shown
that Rab22a wild-type and the positive mutant colocalize
with the CI-M6PR [12]. Moreover, we have observed that
overexpression of these proteins causes a redistribution of
the receptor from a perinuclear localization to large Rab22apositive vesicles in the periphery of the cell. As expected
under control conditions [19], most of the CI-M6PR was
found associated with the TGN with very scarce presence in
early endosomes in GFP-expressing cells (Figs. 6A–C). In
contrast, in cells expressing GFP-Rab22a (WT and Q64L),
triple colocalization between Rab proteins, CI-M6PR, and a
5-min uptake of Rh–dextran indicates that most CI-M6PRcontaining vesicles in the periphery of the cell were loaded
with the early endocytic marker (Figs. 6D–I). Similar
observation was obtained when cells were labeled with an
anti-EEA1 antibody (data not shown). These results indicate
that overexpression of wild-type Rab22a and the positive
mutant alter the normal TGN distribution of the CI-M6PR in
CHO cells.
The extensive redistribution of the CI-M6PR to endosomes could be due to an alteration of the TGN that under
some conditions (e.g., Brefeldin A treatment) mixes with
endosomes [20]. To address this possibility, the distribution
of syntaxin 6—a SNARE protein involved in multiple
membrane fusion events, which associates with the TGN
[21]—was analyzed in cells expressing Rab22a wild type
and the mutants. Overexpression of Rab22a WT or the
positive mutant did not cause any alteration in the
perinuclear distribution of syntaxin 6 (Figs. 7A–F). No
colocalization between Rab22a WT or the positive mutant
with the SNARE protein was observed. Even in the
perinuclear region, careful confocal analysis of several
images showed no colocalization between these proteins
(top insets, Figs. 7C and F). The negative mutant did not
affect the distribution of syntaxin 6. However, this mutant
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Fig. 4. Overexpression of Rab22a WT and the positive and negative mutants does not alter the lysosomal distribution of cathepsin D. Stable CHO cell lines
expressing Rab22a WT (top row), Rab22a Q64L (middle row), or Rab22a S19N (bottom row) were incubated with 1 mg/ml rhodamine dextran for 1 h at 378C.
The marker was chased to lysosomes by a 3-h incubation at 378C. The cells were fixed and cathepsin D was immunolocalized. Images were recorded in a
Nikon inverted microscope with appropriate filters. Fluorescence of GFP (left column), lysosomal marker (lysosomes column), cathepsin D (cathepsin D
column), and superposition of the three channels (overlap column) are shown. In all cells, cathepsin D was found in vesicles containing Rh–dextran chased to
lysosomes. However, only Rab22a Q64L was present in vesicles containing cathepsin D (E–H). Scale bars = 7 Am.
was enriched in a perinuclear region overlapping the
syntaxin-6-labeled region (Figs. 7G–I). As an additional
control using live cells, the integrity of the distal Golgi
cistern was assessed by cotransfecting CHO cells with
pECFP-rab22a and pEYFP-GalNac-T1-27, a protein that is
retained in the distal Golgi [22]. Confocal microscopy of
unfixed cells showed that overexpression of Rab22a WT did
not alter the distribution of GalNac-T1-27 and no colocalization between the two proteins was observed (data not
shown). To assess that overexpression of Rab22a specifically redistributes CI-M6PR without affecting other Golgi
markers, triple labeling of the receptor, GalNac-T, and
Rab22a was performed in a cell line expressing myc-tagged
GalNac-T [15]. In these cells, overexpression of Rab22a
WT and Q64L caused a redistribution of CI-M6PR to the
periphery of the cells, while GalNac-T remained perinuclear
(Figs. 7J–O).
Another explanation for the accumulation of the CIM6PR in endosomes is that Rab22a may delay the transport
of the receptor from endosomes to the Golgi apparatus. To
directly assess this possibility, we searched for ligands that
traverse this pathway. Several lipids, such as the GM1ganglioside, are known to travel through the retrograde
pathway back to the Golgi apparatus [23]. Cholera toxin that
binds GM1 has been used to visualize transport of this
ganglioside from endosomes to the Golgi apparatus.
Unfortunately, CHO cells do not synthesize GM1. Therefore, a CHO cell line that express two transferases that are
required to generate GM1 [15] was used. This cell line was
transiently transfected with Rab22a WT and the mutants and
the kinetics of arrival of cholera toxin to the Golgi region
was studied. The Golgi apparatus was labeled with an antimyc antibody because GalNac-T, which is a distal Golgi
resident protein (see Figs. 7J–O), was myc-tagged. As
shown in Fig. 8, cholera toxin was efficiently incorporated
in these cells. After 10 min of internalization, the toxin
colocalized with Rab22a WT and Rab22a Q64L but not
with the Golgi marker (Figs. 8A–D and E–H). After 1 h of
internalization, most of the toxin colocalized with GalNacT-myc in a perinuclear region in untransfected cells (observe
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
347
Fig. 5. Overexpression of Rab22a wild-type and the positive and negative mutants does not alter the digestion of BSA, LDL, or EGF. (A) To measure LDL
degradation, stable CHO cell lines expressing GFP alone (vector), or as chimera with Rab22a wild-type (Rab22a WT), the positive mutant (Rab22a Q64L) or the
negative mutant (Rab22a S19N) was incubated with15 Ag/ml 125I LDL 2 h at 48C. The cell monolayers were washed and incubated 6 h at 378C. The media were
collected and trichloroacetic acid soluble radioactivity in all the samples was determined. To compare different experiments performed with different 125I LDL
preparations, the data were normalized by dividing the values by the average digestion measured for all the cell types within each experiment (range 27–48%).
(B) In order to follow BSA degradation, CHO were allowed to internalize 125I BSA for 1 h at 208C. The cells were then incubated 30 min at 378 C. At the end of
the incubation, the cells were solubilized in PBS containing 1% Triton X-100. Trichloroacetic acid soluble radioactivity was measured in all the samples. The
data were normalized by dividing the values by the average digestion measured for all the cell types within each experiment (range 20–29%). (C) To assess the
effect of Rab22a on EGF digestion, CHO cells expressing human EGF receptor were transiently transfected with Rab22a WT.GFP and Rab22a S19N.GFP.
Twenty-four hours after transfection, the cells were incubated for 1 h at 378C with 400 ng/ml of rhodamine-labeled EGF. After several washes, the cells were
chased for 0 or 3 h and fixed. The amount of rhodamine fluorescence per cell was quantified in at least 20 transfected and untransfected cells for each condition by
using the MetaMorph. Values represent the mean and SEM of 5, 4, and 2 experiments performed for LDL, BSA, and EGF digestion, respectively. No significant
differences were observed among the groups in A and B or between transfected and untransfected cells in C (one-way ANOVA, P N 0.05).
cells negative for Rab22a proteins in Figs. 9A–H) or cells
expressing the negative mutant (Figs. 9I–L). In contrast,
overexpression of both wild-type Rab22a and the positive
mutant strongly delayed the arrival of the toxin to the Golgi
(observe cells positive for Rab22a proteins in Figs. 9A–H).
In these cells, the toxin was detected in large Rab22apositive vesicles, segregated from the Golgi marker even
after 2 h of internalization (data not shown). A quantitative
analysis of the experiments confirmed that at early times of
internalization, most of the toxin was not present in the
Golgi in transfected and untransfected cells (Fig. 10A).
However, after 1 h of internalization, most of the toxin had
arrived to the Golgi area, except in cells expressing Rab22a
WT or Rab22a Q64L that significantly delayed the transport
to the perinuclear area (Fig. 10B).
These results indicate that overexpression of Rab22a
wild-type or its positive mutant delays the transport from
endosomes to the Golgi apparatus causing the retention in
endosomes of molecules that normally follow this pathway.
Discussion
Rabs are central protagonists in the mechanism of
membrane trafficking [24]. This large family of small
GTPases possesses a series of unique and remarkable
Fig. 6. Rab22a colocalizes with the CI-M6PR in early endosomes. Stable CHO cell lines expressing GFP, Rab22a WT, or Rab22a Q64L were incubated with
rhodamine dextran (Rh–dextran in blue to match Fig. 7) for 5 min at 378C and fixed. The cells were then immunolabeled with an antibody anti-CI-M6PR (CIM6PR). CI-M6PR was present in a perinuclear region in GFP-expressing cells with very scarce colocalization with Rh–dextran (A–C). In contrast, observe that
many CI-M6PR vesicles in the periphery of the cell contained Rh–dextran in WT (D–F)- and Q64L (G–I)-expressing cells. Scale bars = 7 Am. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
Fig. 7. Rab22a WT and the positive and negative mutants do not affect the distribution of TGN markers. Stable CHO cell lines expressing Rab22a WT, Rab22a
Q64L, or Rab22a S19N were fixed and immunolabeled with an anti-syntaxin-6 antibody. Despite the presence of large vesicles in Rab22a WT (A–C)- and
Rab22a Q64L (D–F)-expressing cells, syntaxin 6 maintained a normal perinuclear distribution (the corresponding region of an untransfected cell is shown in
the bottom insets in panels A–C). Some overlapping with Rab22a proteins was observed in this region. However, careful analysis of the cells by confocal
microscopy showed very little colocalization (top insets in C and F). The negative mutant showed its characteristic cytosolic distribution and enrichment in a
perinuclear region that overlapped with syntaxin 6 (G–I). To assess the differential effect of Rab22a on the CI-M6PR and other Golgi marker, CHO cells that
express GalNac-T-myc were transiently transfected with Rab22a WT (J–L) or Rab22a Q64L (M–O). The cells were fixed and immunolabeled with an anti-CIM6PR (CI-M6PR) and with an anti-myc antibody (GalNac-T-myc). Notice the good colocalization between of Rab22a (WT and Q64) with the CI-M6PR and
the lack of colocalization with GalNac-T (insets J–O). It is also evident the perinuclear distribution of CI-M6PR is affected in Rab22a transfected cells, whereas
the distribution of GalNac-T is not disturbed. Scale bars = 7 Am.
properties that are crucial for membrane recognition and
fusion. In addition, they are key factors for the differentiation and segregation of domains in the membranes
limiting several organelles [2,25]. Rabs switch between two
conformations with different overall 3D structure upon
binding to GDP or GTP and these two conformations
interact with different proteins [26]. The active form of Rab
proteins binds a series of multiple domain effectors that
participate in different steps of transport [27]. Rabs are
prenylated at their carboxy terminal region [28]. However,
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
349
Fig. 8. Cholera toxin accumulates in Rab22a WT- and Rab22a Q64L-positive vesicles at early times after internalization. CHO cells expressing transferases
required for synthesizing GM1 were transiently transfected with pEGFP-rab22a WT (top row), pEGFP-rab22a Q64L (middle row), or pEGFP-rab22a S19N
(bottom row). Twenty-four hours later, the cells were incubated with 1 Ag/ml cholera toxin conjugated to Alexa Fluor 594 for 1 h at 48C. After extensive
washing, the cells were incubated for 10 min at 378C and fixed. One of the transferases, which is a distal Golgi enzyme, carried a myc tag. Hence, the Golgi
apparatus was visualized with an anti-myc antibody and an Alexa Fluor 350-labeled secondary antibody. Notice that at this early time of internalization, in
Rab22a-transfected and untransfected cells (GFP fluorescence is shown in the left column), the cholera toxin (cholera toxin column) was not present in the
Golgi apparatus (GalNac-T-myc column). Superposition of the three channels is shown in the overlap column. In cells expressing Rab22a WT and Rab22a
Q64L, the toxin showed a strong colocalization with the fluorescent proteins (D and H). Scale bars = 7 Am.
they can be extracted from membranes by GDI, a protein
that interacts with Rabs in the GDP-bound conformation.
Therefore, these proteins can cycle between the cytoplasm
and the membranes. Despite the possibility of reversibly
detaching from the membranes, Rabs have very welldefined localizations along the endocytic and exocytic
pathways. Indeed they are used as markers for the different
compartments in these pathways. The mechanism that
provides the specificity of interaction of each Rab with a
specific compartment is still not well understood.
Our experiments show that Rab22a extensively colocalizes with Rab5, confirming previous observations from
our laboratory and others that indicate that this small
GTPase is present in early endosomes. Overexpression of
the protein does not block transport of endocytosed material
to lysosomes, although a delay was observed with the
positive mutant. In our system, no significant changes in the
degradation of proteins internalized by fluid phase (BSA) or
receptor-mediated (LDL) endocytosis was observed. Kauppi
et al. [14] have reported that overexpression of wild-type
Rab22a and two mutants (Rab22a Q64L and Rab22a S19N)
in Hep2 cells significantly increases the percentage of cells
presenting vesicles containing EGF after 3 h of chase. The
authors conclude that Rab22a (wild-type and the positive
and negative mutants in a lesser extent) inhibits EGF
degradation in these cells. We have performed similar
experiments in CHO cells expressing EGF receptor. No
differences were observed between untransfected cells and
cells transfected with pEGFP-rab22a (WT or S19N),
indicating that these proteins do not disrupt lysosomal
digestion of endocytosed proteins in CHO cells.
Both wild-type Rab22a and the positive mutant caused a
striking redistribution of CI-M6PR from a Golgi perinuclear
region to peripheral large vesicles labeled with Rab22a.
Triple colocalization assays with early endosomal markers
(5 min uptake of Rh–dextran and EEA1) showed that many
of these vesicles were early endosomes. In contrast,
expression of Rab22a (WT and mutants) did not alter the
perinuclear localization of syntaxin 6, a protein present in
the TGN. In a previous paper, we have reported that Rab22a
WT does not affect the distribution of two other Golgi
markers (membrin and BODIPY-TR ceramide). We have
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Fig. 9. Overexpression of Rab22a WT and Rab22a Q64L delays the transport of cholera toxin to the Golgi apparatus. In the same experiment described in Fig.
8, cholera toxin was allowed to progress to the Golgi apparatus by a subsequent incubation for 1 h at 378C before fixation. The Golgi resident transferase was
immunolabeled with an anti-myc antibody and an Alexa Fluor 350-labeled secondary antibody. Notice that in all untransfected cells and cells expressing
Rab22a S19N (GFP fluorescence is shown in the left column), cholera toxin (cholera toxin column) has arrived to the Golgi apparatus (GalNac-T-myc column).
In contrast, in cells overexpressing Rab22a WT and Rab22a Q64L, the majority of the toxin is still in the periphery colocalizing with Rab22a-positive
structures. Superposition of the three channels is shown in the overlap column. Scale bars = 7 Am.
extended this observation to the positive mutant (data not
shown). To show that the effect is specific for the CI-M6PR,
we performed triple localization studies with this receptor,
GalNacT-myc (a Golgi-associated protein) and Rab22a (WT
and Q64L). The results show that only the CI-M6PR is
redistributed in transfected cells. Despite the altered
distribution of the CI-M6PR, cathepsin D (a lysosomal
enzyme that binds this receptor) localized in lysosomes.
Similar results have been reported by another laboratory
[14]. These authors showed that the transport of overexpressed aspartylglucosaminidase—another enzyme that is
recognized by the CI-M6PR—from the Golgi apparatus to
lysosomes is not affected in BHK-21 cells cotransfected
with wild-type Rab22a. If Rab22a does not hamper the
transport out of the Golgi apparatus, it may affect the
reverse path from endosomes to the Golgi. To assess this
possibility directly, we searched for ligands that are transported from the plasma membrane to the Golgi apparatus
via the endosomal compartment. The GM1 ganglioside
follows this pathway, and cholera toxin, a natural ligand for
the lipid, has been used to visualize the intracellular
transport in living cells [23]. A stable CHO cell line that
produces GM1 [15] was transfected with Rab22a WT and
the mutants. In untransfected cells, most of the toxin bound
to GM1 in the plasma membrane was efficiently transported
to the Golgi apparatus after 1 h of internalization. In
contrast, a remarkable inhibition of the transport from
endosomes to the Golgi apparatus was observed in both
Rab22a WT- and Rab22a Q64L-expressing cells. Kauppi
et al. [14] have reported that high-level expression of
Rab22a WT or Rab22a Q64L causes vesiculation of the
Golgi apparatus in HeLa and occasionally in BHK cells. We
have not observed disruption of the Golgi architecture in
CHO cells at any level of expression using different Golgi
markers. However, the prominent effect of Rab22a in the
trafficking between endosomes and the Golgi cisternae may
cause alterations in this organelle in other cell lines. It is
worth mentioning that Rab22b has been implicated in the
transport from the trans-Golgi to endocytic compartments in
HeLa cells and oligodendrocytes [29].
Overexpression of dominant positive mutant of Rab22a
causes a dramatic enlargement of endosomes. However,
R. Mesa et al. / Experimental Cell Research 304 (2005) 339–353
Fig. 10. Delayed transport of cholera toxin to the Golgi by overexpression
of Rab22a WT and Rab22a Q64L, a quantitative analysis. At least 40
transfected and untransfected cells for each condition were processed by
using the MetaMorph software to measure the percentage of cholera toxin
not associated with the Golgi marker. (A) At 10 min after the internalization, about 80% of the toxin was not in the Golgi apparatus and there
were not significant differences between any of the cells analyzed. (B) After
1 h chase, only 20% of the cholera toxin was not in the Golgi apparatus in
untransfected cells or in cells overexpressing the negative mutant (S19); in
contrast, about 60% of the toxin had not yet arrived to the Golgi in cells
expressing Rab22a WT (WT) or the positive mutant (Q64L). Values
represent the mean and SEM of at least 40 cells recorded in two
independent experiments. Asterisks, significant differences ( P b 0.001)
with respect to the corresponding untransfected cells (Student’s t test for
unpaired observations).
different to what was observed with the wild-type protein,
the positive mutant colocalizes with both endosomal and
lysosomal markers. The kinetics studies performed indicate
that Rab22a Q64L does not promote the mixing of all
endocytic compartments. The presence of this Rab in
vesicles containing lysosomal markers is better explained
by a mislocation of the mutant to lysosomes. The lack of
hydrolysis of GTP may prevent the mutant to switch from a
GTP-bound form to the GDP-bound form that can cycle to
the cytosol in complex with GDI. Kauppi et al. [14] have
also observed that Rab22 Q64L colocalizes with lysosomal
markers. However, these authors interpret their results as a
mixing of compartments because they report colocalization
of EEA1 (an early endosome marker) and Lamp1 (a late
endosome/lysosome marker) in cells expressing Rab22a
Q64L. An alternative explanation would be that because
Rab22a Q64L tightly binds EEA1, the positive mutant
causes mislocation of this protein to lysosomes. Positive
mutants of several Rabs cause dramatic enlargement of
several compartments most likely by promoting homotypic
fusion between vesicles and tubules. One of the bestcharacterized positive Rab mutants is Rab5 Q79L. In cells
overexpressing Rab5 Q79L, large vesicles decorated with
the protein containing lamp2 and CI-M6PR have been
observed [30]. Similar to what is observed with Rab22a
Q64L, internalized ligands remained for long period of
chase in Rab5 Q79L-positive vesicles [30,31]. However, the
uptake and recycling of transferrin [32] and h2-adrenergic
receptor [30] are not affected, suggesting that the endocytic
351
pathway is not blocked despite the dramatic morphological
changes caused by the mutant.
Rab proteins are well-known molecular switches activating homotypic fusion between vesicles. Rabs are probably
needed in both membranes to trigger tethering before fusion
[33]. Heterotypic fusion may result from the interaction
between membranes containing different Rabs that share the
same effectors, such as Rab5 and Rab4 [34]. However, Rabs
are not the only set of proteins determining fusion
specificity; the SNARE membrane proteins are also an
important part of the membrane recognition and fusion
process [35]. Therefore, mislocalization of Rab-positive
mutants may promote homotypic fusion and enlargement of
all compartments where they are attached, but heterotypic
fusion with other compartments may be limited because of
the incompatibility of the SNAREs present in the membranes. Rab22a Q64L may locate to endosomes and
lysosomes, but both organelles may keep their identity
because they do not carry compatible SNARE cognates.
No major effects were observed with Rab22a S19N. This
mutant has a reduced affinity for GTP and causes a decrease
in the steady state accumulation of a fluid phase marker
[12]. However, we have not observed any effect on the
lysosomal function or in the trafficking between endosomes
and the Golgi apparatus. A very mild phenotype for Rab22a
S19N has also been reported by another laboratory [14].
Most Rabs with reduced affinity for GTP are dominantnegative factors because they interfere with the exchange
factor required for activation of the endogenous protein
[36]. Rab22a S19N may not efficiently inhibit the exchange
factor of Rab22a. Alternatively, the endogenous Rab22a
may have a minor role in the endocytic transport of the cell
lines where the effect of the mutant was assessed. Although
Rab22a S19N was not useful as a dominant-negative
mutant, it clearly showed that only the GTP-bound form
of Rab22a modulates the transport from endosomes to the
Golgi apparatus.
In a very recent paper, Rab22a has been involved in the
recycling of MHCI molecules from endosomes to the
plasma membrane in HeLa cells [13]. These authors propose
that Rab22a functions in the recycling of specific molecules
such as MHCI from a compartment occurring downstream
the site of merging of the clathrin-dependent and independent endocytic pathways. It is worth noting that
some proteins are transported to the TGN from recycling
endosomes [10,19,37] and that protein recycling and transport to the TGN share some common regulators [38].
However, we have not observed prominent Rab22a localization in the CHO cell recycling compartment even at very
low expression levels (Magadan and Mayorga, unpublished
observations). Further experimental work will be required to
unveil the relationship between the function of Rab22a in
MHCI recycling in HeLa cells and the effect reported by us
on the endosome-to-TGN pathway in CHO cells.
The results that we have presented indicate that Rab22a
regulates transport between endosomes and the Golgi
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apparatus, probably by promoting fusion between endosomes and preventing the formation of isolated microdomains in the membranes that may be necessary for
targeting of vesicles to the Golgi apparatus.
Acknowledgments
We thank A. Medero for excellent technical assistance;
S. Kornfeld and W. Brown for antibodies; B. van Deurs, H.
Maccioni, and S. Pfeffer for plasmids; and L. Daniotti and
J. Pessin for special CHO cell lines. We would like to
thank M.I. Colombo and C. Tomes for critically reading of
the manuscript. This work was partly supported by an
International Research Scholar Award from the Howard
Hughes Medical Institute and by a grant from Agencia
Nacional de Promoción de la Ciencia y la Tecnologı́a,
Argentina. RM received support from The International
Union of Biochemistry and Molecular Biology and
Fundación Antorchas for a training visit to Philip Stahl’s
Laboratory (St. Louis, USA).
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