1
Department of Microbiology and Immunology, LSU Health Sciences Center,
Shreveport, LA 71130, USA
2
Department of Botany, Kyoto University, Kyoto 606-8502 Japan
3
Department of Biology, University of Arkansas at Little Rock, Little Rock, AR
72204, USA
*
Author for correspondence (e-mail:
jmbush{at}ualr.edu
)
Accepted May 9, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dictyostelium, DdRab11, Contractile vacuole
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The secretory vesicular pathway originates at the endoplasmic reticulum and
passes through the Golgi into lysosomes, secretory granules or the
extracellular environment via exocytosis. At least four members of the Rab
GTPase family associate with the secretory pathway, including Rab1 (ER to
Golgi), Rab3 (exocytosis), Rab6 (Golgi) and Rab11 (secretory granules and
endosome; Nuoffer and Balch,
1994; Green et al.,
1997
; Ren et al.,
1998
). The other extensively
studied vesicle transport pathway is the endocytic pathway by which material
from the extracellular environment of the cell can be internalized
(endocytosed) and transported through early and late endosomal organelles
arriving in the lysosomes (site of intracellular digestion; Nuoffer and Balch,
1994
). At least five members
of the Rab family have been localized to this pathway, including Rab4, 5 and
11 (early endosomes) plus Rab7 and Rab9 (late endosomes; Nuoffer and Balch,
1994
; Green et al.,
1997
; Ren et al.,
1998
).
The process of membrane and protein transport has been extensively studied
in many systems, including the simple eukaryote, Dictyostelium
discoideum. This genetically tractable amoeba shares similarity with
human leukocytes in terms of a prominent endocytic pathway and a highly
phagocytic nature (reviewed by Rupper and Cardelli,
2001; Maniak,
2001
). Extracellular fluid
phase material can be endocytosed via a variety of different processes,
including micropinocytosis and macropinocytosis. As observed in mammalian
cells, fluid is transported through endosomes into acidic lysosome-like
vesicles in D. discoideum (Rupper and Cardelli,
2001
; Maniak,
2001
). Uniquely in D.
discoideum, cargo is then transported from lysosomes to larger neutral
post-lysosome-like compartments before egestion (Rupper and Cardelli,
2001
; Maniak,
2001
). The D.
discoideum endocytic pathway also differs from mammalian cells in the
apparent lack of rapid fluid recycling from early endosomal compartments back
to the cell surface, although plasma membrane proteins are rapidly recycled
(Neuhaus and Soldati, 2000
).
In D. discoideum, endocytic fluid phase trafficking is linear and
extracellular cargo moves through all of the endocytic compartments before
leaving the cell (Maniak,
2001
).
Like other protozoans, D. discoideum has a contractile vacuole
(CV) complex that serves to excrete cytoplasmic water and insures survival in
a hypo-osmotic environment. The CV complex is enriched in membrane-associated
alkaline phosphatase, calmodulin (Ca2+-binding protein) and
vacuolar proton pumps (V-H-(+) ATPase; Nolta and Steck,
1994). This complex has a
bipartite morphology that consists of a bladderlike pump vacuole and an
associated tubular spongiomal network (Nolta and Steck,
1994
). The CV is the site of
water collection in the cell, and excess water is excreted using a
myosin-driven contraction that forces water from the vacuole into the
extracellular medium (Nolta and Steck,
1994
). In contrast to the CV
bladder, the spongiomal network contains little protein other than V-H(+)
ATPase, which has been predicted to catalyze the primary energy reactions
needed to pump water to the vacuole (Heuser et al.,
1993
; Nolta and Steck,
1994
).
In an earlier screen, we isolated 20 members of the Ras superfamily of
GTPases from a D. discoideum cDNA library (Bush et al.,
1993; Daniels et al.,
1994
; Bush et al.,
1994
; Hall,
1993
; Bush and Cardelli,
1995
). Several of the Rab-like
GTPases have been further characterized, including two Rab GTPases, RabD (a
Rab4 homolog) and Rab7, which are between 70 and 80% identical at the amino
acid level to their human homologs (Bush et al.,
1996
; Buczynski et al.,
1997
).
All reported Rab GTPases are thought to function in the processes of
eukaryotic vesicular transport and fusion between intracellular compartments
or in exocytosis from the cell (Mellman,
1994). Our observation that
RabD was localized to the CV complex and endo-lysosomes suggested that an
uncharacterized membrane transport system connecting the endo-lysosomal and CV
system exists in D. discoideum (Bush et al.,
1994
). Supporting this idea, we
have found that the RabD regulates not only the function of the CV complex but
also the localization of two associated CV complex marker proteins, calmodulin
and vacuolar ATPase (V-H(+)ATPase; Bush et al.,
1996
). Additional experiments
revealed that the RabD was also involved in the regulation of the early
endocytic pathway in D. discoideum (Bush et al.,
1994
). In this report, we
describe the cloning and analysis of a Dictyostelium DdRab11
homologue and demonstrate that this Rab protein, like RabD, also regulates the
structure and function of the CV complex.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning cDNAs
A standard plaque lift (Sambrook et al.,
1989) was used to screen a
recombinant cDNA library constructed in lamda gt11, using mRNA prepared from
cells that had developed for 4 hours (Bush et al.,
1993
). Probe design,
hybridizations, washing conditions and PCR sequencing of the DNA were as
previously described (Bush et al.,
1993
).
Antibody production
The DdRab11 cDNA was ligated directionally into the pRSET bacterial
expression vector and used to generate affinity-purified protein suitable for
immunization of New Zealand male rabbits (Cocalico Biologicals). Specific
DdRab11 antibodies were isolated and tested for crossreactivity to other
Dictyostelium Rab proteins (DdRab2, DdRabD, and DdRab7) by Western
Blot analysis (Bush et al.,
1994).
Subcellular fractionation
Membrane fractionation of Ax4 cells was performed as described
previously (Bush et al., 1994).
The resulting crude and high-resolution membrane fractions were subjected to
SDS-PAGE and western blot analysis, as described below in the western blot
analysis section. Magnetic fractionation of Ax4 cells has also been
detailed before (Rodrigeuz-Paris et al.,
1993
).
Western blot analysis
Proteins were separated by SDS-PAGE and transferred to nitrocellulose.
Blots were then probed with Rabbit serum primary antibodies to DdRab11 (Bush
et al., 1994) washed, incubated
with goat anti-rabbit secondary antibody conjugated to alkaline phosphatase
(Sigma) and visualized using Sigma FAST tablets (Sigma).
Immunofluorescence microscopy
Both wild-type and mutant cells were allowed to settled onto plastic
coverslips, fixed, and probed with primary and secondary antibodies (Bush et
al., 1994). Stained cells were
photographed using an Olympus model BH-2 fluorescence microscope and Kodak
T-Max 400 speed film.
Molecular techniques
The DdRab11 cDNA was subjected to oligonucleotide-mediated site-directed
mutagenesis in order to change a key amino acid asparagine (N) to isoleucine
(I) at amino acid position 125 in the predicted open reading frame of the
DdRab11 gene. The mutated cDNA was then sequenced to confirm the single change
in the DdRab11 protein. The mutant DdRab11 cDNA was placed into a
Dictyostelium expression vector pHA80 and wild-type cells were
transformed. Proteins expressed from this vector are constitutively expressed
as fusions inframe with an N-terminal HA amino acid tagged epitope, detectable
by a monoclonal antibody (Bush et al.,
1996).
GFP DdRab11 expression
The complete Dictyostelium DdRab11-coding region cDNA (Bush et
al., 1994) was modified with
the addition of BamHI recognition site DNA at the end of the DdRab11
cDNA and the addition of XbaI SacI recognition site DNA at
the beginning of the DdRAB cDNA using oligonucleotide mediated PCR. The
resulting PCR product was ligated into the TA vector (Invitrogen) and
sequenced for both errors and confirmation for the presence of the N-terminal
XbaI SacI sites and C-terminal BamHI sites. The
DdRab11 PCR TA product was then digested with BamHI and
XbaI, purified and ligated into the pDXA-HC vector previously cut by
BamHI and XbaI (Manstein et al.,
1995
). This new construct
pDdRab11DXA-HC was then sequenced for errors before the next ligation
step.
DNA encoding green fluorescent protein (GFP) was modified by the addition
of KpnI N-terminal and SacI C-terminal via oligonucleotide
mediated PCR. The resulting PCR product was subcloned in the TA vector and
sequenced for errors and confirmation of KpnI and SacI
recognition site presence. The modified GFP DNA was then digested with
KpnI and SacI and ligated into the pDXA-HC DdRab11 vector,
previously cut with SacI and KpnI, and purified. This vector
construct DdRab11 GFP was sequenced for errors and reading frame conformation.
The DdRab11 GFP plasmid and the pREP plasmid (ORF helper plasmid; Manstein et
al., 1995), were then
co-transformed into Dictyostelium cells as previously described (Bush
et al., 1996
) and properly
expressing cells selected via use of G418 antibiotic selection.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Southern blot analysis using the DdRab11 cDNA as a probe under low stringency hybridization conditions revealed that Dictyostelium DdRab11 was related in sequence to at least one other gene (results not shown). Northern blot analysis using the same cDNA probe detected a single message at 0.8 kb whose steady state levels remained constant throughout the developmental cycle of this organism (results not shown).
Western blot analysis using an affinity purified rabbit antibody to DdRab11 revealed a single protein of 24 kDa in total cellular proteins consistent with the predicted molecular weight for this protein (Fig. 2); this band was not observed when preimmune serum was used. In addition, the DdRab11 antibody did not crossreact with either Rab7 or RabD.
|
Western blot analysis revealed that 95% of the DdRab11 protein was
associated with total cell membranes after centrifugation of cell homogenates.
Next, subcellular fractionation experiments were carried out to determine
which membrane system DdRab11 associated with. First, post-nuclear
supernatants were fractionated on percoll gradients and the separated
fractions were subjected to SDS-PAGE and western blot analysis. Marker enzyme
activities for both the light fractions and lysosomal fractions were
determined, and corresponding fractions were grouped as shown in
Fig. 2A,B. DdRab11 was found to
be mainly associated with buoyant membrane fractions that contained endosomes
and elements of the CV, while relatively little DdRab11 was found in the dense
lysosomal fractions of the gradient (Fig.
2). In contrast, RabD distributed in two peaks on the Percoll
gradients. The majority of RabD distributed in the region of the gradient
containing the CV membranes while the remainder was found in the region
containing lysosomes. This distribution is consistent with the known
association of RabD with both of these membrane systems (Bush et al.,
1994; Bush et al.,
1996
).
The next fractionation scheme involved both magnetic separation to purify
lysosomes (Rodrigeuz-Paris et al.,
1993) and sucrose density
gradients to purify the CV system of membranes. Lysosomal fractions were
purified from cells pulsed with colloidal iron for 15 minutes and chased for
30 minutes. Western blot analysis of these lysosomal and CV preparations
revealed that DdRab11 was enriched fivefold in the CV relative to levels found
in the PNS, while no detectable DdRab11 was observed in purified lysosomes
(Fig. 2D). Consistent with
previously published data, RabD was enriched in both the CV and in lysosomes
(Fig. 2C). Taken together,
these fractionation data strongly suggest that DdRab11 is highly enriched in
the CV system. This is the second Rab from this organism to be shown to be
associated with the CV complex, the other being the Rab4-like RabD GTPase
(Bush et al., 1994
).
Microscopic approaches were taken to confirm that DdRab11 associates with
the CV. It has been demonstrated that calmodulin, V-H(+)ATPase, and RabD are
localized to the CV in Dictyostelium (Heuser et al.,
1993; Zhu et al.,
1993
; Bush et al.,
1994
). Immunofluorescence
microscopy (IF) was used to determine whether DdRab11 co-localized with either
of these established CV marker antigens.
Fig. 3 shows the results of
these co-localization studies, in which detergent permeablized cells were
incubated with mouse monoclonal antibodies to the 100 kDa V-H(+) vacuolar
ATPase subunit and affinity-purified rabbit polyclonal antiserum to DdRab11 or
RabD. Cells were then washed and incubated with secondary antibodies coupled
with rhodamine (goat anti-rabbit) and fluorescein (goat anti-mouse). DdRab11
(Fig. 3A) was associated with a
reticular network of membranes that contained the proton pump
(Fig. 3B); DdRab11 also
co-localized with RabD. In addition, as previously reported, RabD
(Fig. 3C) and the proton pump
(Fig. 3D) co-localized to this
reticular network (Bush et al.,
1994
). Thus, we conclude that
DdRab11, RabD and the proton pump are all found in the same membrane system,
namely the CV network.
|
To rule out the slight possibility that the DdRab11 antibody was crossreacting with a related Rab11-like protein, we examined the distribution of DdRab11 tagged at the N terminus with GFP. Fig. 4 shows that GFP-DdRab11 distributes in a reticular network of membranes (Fig. 4A,B) that appeared identical to that of the proton pump detected by immunofluorescence microscopy (Fig. 4C-E).
|
GFP-tagged DdRab11 associates with the CV network, fusing bladders
and bladders undergoing expulsion of water
Fig. 5 represents an example
of real time movie images of cells expressing GFP-DdRab11 in a photographic
sequence lasting for 12 seconds. Each image of this sequence (images 1 through
12) represents 1 second of elapsed time. As can be seen in these images,
GFP-DdRab11 is associated with a very dynamic CV network connected to
vacuoles, comparable with the images observed previously (Gabriel et al.,
1999). A very prominent
vacuole at the lower bottom (6 o'clock position) can be seen in the images
labeled 1 to 12 to be undergoing the process of water expulsion. Notice that
DdRab11 remained associated with this structure even after expulsion (image 11
and 12). This vacuole also apparently began to lose connection with the CV
complex just before expulsion (images 5-10), as has been previously suggested
(Heuser et al., 1993
).
|
Fig. 6 is another example of movie images of GFP DdRab11 in a photographic sequence lasting for 12 seconds (images1 through 12) with each image representing about 1 second of elapsed time. Images 3 and 4 of this figure document an apparent homotypic fusion event between two adjacent CV elements. In image 3, the white arrowhead indicates the area of fusion between two CV elements beginning in image 2 and completed in image 4. The size of this large vacuole appeared to decrease in size, possibly owing to the formation of a second adjacent CV as seen in images 8-11.
|
Images in Fig. 6 also show the formation of a GFP-Rab11-positive CV bladder. Beginning in image 1, the GFP-DdRab11 stained CV network, as marked by the dashed circle, shows a region of the network that is transformed into a CV bladder. Moving to image 4, one sees the apparent formation of an irregular round `filled in' structure that slowly rounds into a more recognizable bladder by images 10 through 12. The size of the bladder also increases during this time. This rounding and size increase probably corresponds to the filling of water into this structure.
Finally, as seen in image 1 of Fig. 6, two CV bladders (marked with double arrows) are beginning the process of water expulsion. Following these CVs through the first seven images demonstrates that the network is released from them, followed by an almost simultaneous water expulsion event (see double arrows). The GFP DdRab11-positive membrane remnants then seem to form small vesicles, most likely at the cell surface, which may rejoin the network.
Different osmotic conditions differentially effect the subcellular
localization of DdRab11 and other CV proteins
RabD, DdRab11, calmodulin and the proton pump all co-localize to the CV
network of membranes in cells in normal growth medium (HL5). To determine if
the intracellular location of any of these proteins changed in cells
osmotically challenged, cells were collected by centrifugation and resuspend
in hyperosmotic (100 mM sucrose in HL5) or hypoosmotic medium (10 mM phosphate
buffer) for 1 hour. Cells were spotted on coverslips for 10 minutes and then
processed for immunofluoresence microscopy.
Cells exposed to 100 mM sucrose in HL5 accumulated large endosomal vesicles
ringed with Rab7 (Buczynski et al.,
1997). Under these conditions,
DdRab11 remained associated with the reticular network and continued to
co-localize with calmodulin (Fig.
7A,B, respectively). RabD and calmodulin also remained
co-localized in the CV under hyper-osmotic conditions
(Fig. 7C,D, respectively).
Surprisingly, the proton pump 100 kDa subunit no longer co-localized with
DdRab11 in the CV, but instead accumulated in small vesicular structures
(Fig. 7E,F). The vesicles
(denoted by the arrow in Fig.
7F) are not part of the endosomal pathway (Buczynski et al.,
1997
).
|
In cells exposed to hypo-osmotic conditions (100% water), DdRab11 no longer co-localized with the proton pump (Fig. 8C-E). Under these conditions, the proton pump was found to distribute in swollen CV bladders and residual reticular membranes in many cells (Fig. 8 D,E, marked with an arrowhead). By contrast, DdRab11 was distributed in a diffuse punctate pattern with an accumulation of puncta at the cell surface (Fig. 8C, marked with an arrowhead), while RabD remained co-localized with the proton pump (Fig. 8A,B). The focal plane imaged in Fig. 8A,B reveal predominately a network, although other focal planes in this cell contained vesicles like those observed in Fig. 8D.
|
Inhibition of proton pump activity by concanamycin A results in an increase
in endosomal pH and accumulation of swollen endo-lysosomes (Temesvari et al.,
1996). Under these conditions,
the proton pump was found to localize primarily in these large endo-lysosomal
vacuoles (Fig. 9B,C, arrow),
while DdRab11 (Fig. 9A)
distributed in a diffuse punctate pattern with no accumulation of at the cell
surface. RabD also did not co-localize with the proton pump under these
conditions (compare Fig. 9D with
9E, arrows). These results suggest that two Rab GTPases, RabD and
DdRab11, are not unconditionally co-localized with each other or the proton
pump, supporting the hypothesis that the GTPases regulate unique membrane
trafficking steps in different subcompartments of the CV system.
|
Generation of stable cell lines overexpressing DdRab11 N125I
To begin to determine the function of DdRab11 in Dictyostelium, an
asparagine was changed to an isoleucine at amino acid position 125 by
site-directed mutagenesis. This amino acid plays a crucial role in the binding
of GTP and GDP, and comparable mutations in other Rab proteins result in the
formation of proteins that function in a dominant negative manner (Nuoffer and
Balch, 1994).
The mutated cDNA was subcloned behind and inframe with DNA encoding the HA
flu epitope; N-terminal epitope tagging has been shown not to effect the
location and function of this small molecular weight GTPase (Bush et al.,
1994). Expression of dominant
negative DdRab11 (DdRab11N125I) was under the control of the actin 15 promoter
that is constitutively active in axenically growing cells. Wild-type cells
were transformed with this plasmid and several G418 resistant clones were
isolated. Western blot analysis of these clones was performed and the results
showed that transformed cells contained both a 24 kDa wild-type DdRab11
protein as well as a 25 kDa HA-tagged mutated version of the DdRab11 GTPase,
as confirmed by decoration with HA-specific antibodies (J.C. and J.B.,
unpublished). Clones that had at least 2-fold over-expression of the HA-tagged
DdRab11 were then selected for further study.
Morphology and function of the contractile vacuole system is altered
in cells expressing dominant negative DdRab11
Expression of dominant negative form of RabD resulted in the redistribution
of ATPase-positive CV membranes into a patch of vesicles at the cell surface
(Bush et al., 1996).
Furthermore, these mutant cells swell and burst when placed in water,
suggesting that the CV system is no longer functioning normally. Cells
expressing DdRab11N125I were subjected to this `water test'. Exponentially
growing shaking suspension cultures of wild-type and mutant cells were allowed
to attach to a plastic surface for 15 minutes and then the growth medium was
removed, replaced with water and the cells photographed after 15 minutes.
Representative phase-contrast photomicrographs of both wild-type and mutant
cells after 15 minutes in water are shown in
Fig. 10A,B, respectively.
After 15 minutes in water, most wild-type cells
(Fig. 10A) were attached to
the plastic and were amoeboid in shape. However, the mutant cells
(Fig. 10B) were rounded and
detached from the plastic surface. Many mutant cells also had obvious enlarged
and swollen contractile vacuoles (arrowheads). After 2-3 hours, the mutant
cells remained swollen and rounded with some lysis of the cells.
|
To determine whether DdRab11N125I-expressing cell lines demonstrated alterations in the structure of the CV system consistent with altered function, we used indirect immunofluoresence to visualize DdRab11 and the 100 kDa proton pump subunit. Fig. 11 shows representative photomicrographs of both wild-type and mutant cells stained with antibodies to the 100-kDa ATPase protein. The 100 kDa ATPase subunit and in wild-type cells (Fig. 11B) were mainly co-localized as previously shown (Fig. 4). In cells expressing DdRab11N125I, the distribution pattern of the 100 kDa ATPase subunit was strikingly altered. The proton pump protein was found in both bladders and reticular structures, in a pattern similar to control cells; however, the 100 kDa subunit appeared to be concentrated in a thickened and more elaborate reticular network (Fig. 11A). In several cells, enlarged CV bladders were also seen (results not shown). These may represent the previously described large CV bladders that result from homotypic CV fusions (Fig. 6). The prominence of these enlarged CV bladders in the mutant cells suggests a role for DdRab11 in exocytosis, shrinking or fission of these structures.
|
Cells expressing dominant negative DdRab11 appear normal in most but
not all endocytic processes
As described above, DdRab11 associated predominantly with the CV system of
membranes with no significant localization in the endosomal pathway. This
suggests that expression of DdRab11N125I would not affect endocytic processes.
Therefore, it was not surprising that the rates of fluid phase uptake
(Fig. 12; panel endocytosis)
and fluid release (Fig. 12,
top) were similar between control and mutant cell-lines. Surprisingly, cells
expressing DdRab11N125I internalized particles at twice the rate of control
cells (Fig. 12, bottom).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results suggest that DdRab11 may be involved in membrane trafficking important in the proper formation and function of the CV. Supporting this conclusion are the observations, based on a variety of approaches, that DdRab11 is greatly enriched and exclusively found in the contractile vacuolar membrane system. This is now the second Rab GTPase, besides RabD, that is enriched in the contractile vacuole system that appears to regulate membrane flow in and out of this system. Cell lines overexpressing DdRab11N125I protein displayed a functional defect in their water homeostasis, and displayed morphological alterations in their contractile vacuolar membrane structure. Taken together, these results indicate that the CV system needs to regulate membrane flow to maintain its normal morphological status and this flow is regulated by at least two Rab proteins (DdRab11 and RabD).
DdRab11 is associated with contractile vacuoles that are undergoing water expulsion, contractile vacuoles that are newly forming and contractile vacuoles that are undergoing homotypic fusions; DdRab11 may regulate one or more of these processes. Supporting this idea are data that reveal the presence of enlarged contractile vacuolar structures in cells expressing DdRab11N125I. These structures are most likely homotypically fused contractile vacuole bladders that have not undergone proper formation/deformation. These very large contractile vacuole bladders are usually not seen in wild-type cells under the same conditions. In addition, the data from the `water test' on these mutant cells suggests that DdRab11 functions in vesicle expulsion. The mutant cells swell, round up and float off a flat surface. They also have very prominent enlarged contractile vacuoles as seen by light microscopy.
As reported here, DdRab11 is localized almost exclusively in the CV network. In addition, we were not able to detect DdRab11 in magnetically fractionated endosomal/lysosomal membranes (Fig. 2), and endocytosis and efflux were not altered in DdRab11N125I cell lines. However, on rare occasions, we see DdRab11 associated with fluid phase markers (Fig. 4). These data suggest that on unique occasions some interaction between the two compartments takes place and DdRab11 is briefly associated with fused elements of the endosomal and contractile vacuolar systems. Alternatively, DdRab11-containing membranes may associate with the endolysosomal system via formation of autophagosomes.
The role of Rab11 appears diverse, but in many mammalian cell types this
Rab may be involved in the transport of vesicles to the cell surface. For
example, Rab11 is associated with the recycling compartments that contain
transferrin in K562 cells and has been found to regulate recycling through the
pericentriolar recycling endosome in CHO and BHK cells (Green et al.,
1997; Ren et al.,
1998
; Ullrich et al.,
1996
). Besides functioning in
the endosomal system, in gastric parietal cells Rab11 is found in
immunoisolated H+/K+-ATPase-containing tubulovesicles,
and may be involved in the function of these tubulovesicles in apical
regulated vesicle fusion (Urbe et al.,
1993
). Rab11 has also been
shown to be associated with secretory vesicle transport in mammalian cells
(Calhoun et al., 1998
; Calhoun
and Goldenring, 1996
).
The almost exclusive CV localization of DdRab11 in Dictyostelium
would seem to eliminate an important role for this protein in endosomal
recycling. Consistent with this, fluid phase trafficking proceeds in a linear
fashion from small pinosomes and macropinosomes to endosomes, then lysosomes
and finally moving through a large post-lysosomal vesicle population to be
secreted via exocytosis. It has been reported that the contractile vacuolar
and endosomal membrane systems are separate and do not communicate with each
other via vesicle trafficking (Gabriel et al.,
1999; Becker et al.,
1999
), even though RabD and the
proton pump are apparently localized to both systems (Heuser et al.,
1993
; Bush et al.,
1994
). However, it has recently
been demonstrated that internalized plasma membrane proteins are rapidly
recycled back to the cell surface (Neuhaus and Soldati,
2000
) and at least two of
these plasma membrane proteins appear to recycle through the CV network (E.H.
and J.C., unpublished). Furthermore, in a study by the Gerisch group (Gabriel
et al., 1999
), fluorescent
fluid phase markers were used to visualize the endosomal system, and the
authors correctly concluded that no fluid phase was ever found in the CV
network (visualized with GFP-dajumin). Except for the use of Cy-3-tagged cell
surface proteins (Cy-3 can be cleaved in endo-lysosomes), no attempt was made
to analyze the distribution of specific internalized plasma membrane proteins.
One of the functions of DdRab11 therefore could be to regulate the movement of
bladder membranes and perhaps a subset of plasma membrane proteins to the cell
surface from the CV network. In fact, the CV network may be an organelle that
possibly is an evolutionary precursor to the recycling endosomal system in
higher eukaryotic systems. Another possibility could be that as eukaryotic
cellular evolution progressed, the loss of a needed osmoregulation system in
multicellular organisms led to a conservation of the Rab11 function of
directing membrane transport from recycling membrane compartments to the
plasma membrane (endosomal system recycling).
Sheff et al. have reported that in polarized epithelial cells, Rab4 was
enriched in early endosomes while DdRab11 was enriched in recycling endosomes
(Sheff et al., 1999). These
authors report physical separations of the two compartments of the endosomal
system and that the two Rab proteins are involved in the separate functional
endosomal compartments. This physical separation is also seen in
Dictyostelium. RabD (Rab4-like) is localized in endosomal/lysosomal
membranes and contractile vacuolar spongiumal vesicles whereas the DdRab11
protein is associated only with the CV system. This separation is probably
because of the different functional nature of the two Rab proteins. Expression
of dominant negative RabD results in the formation of a patch of CV membrane
at the cell surface and defects in the endosomal system (Bush et al.,
1996
), including a reduction in
the fusion of lysosomes to generate post-lysosomes. By contrast, expression of
DdRab11N125I results in the formation of swollen CV reticular elements with no
obvious changes in the CV system.
Although immunofluorescence microscopy images suggest that RabD and DdRab11
co-localized to the same CV elements, immuno-electron microscopy will be
needed for confirmation. In fact, based on the differential response of these
two Rab proteins to changes in osmolarity, it is possible that they associate
preferentially with different subcompartments of the CV. This type of
association of different Rabs to different subcompartments of the same
organelle has already been observed for mammalian Rab4, Rab5 and hRab11
(Sonnichsen et al., 2000). It
was also intriguing that DdRab11 no longer co-localized with the proton pump
or RabD in cells subjected to hypo-osmotic conditions. This could mean that
Rab11 functions to regulate water efflux under normal conditions but no longer
functions on extreme hypo-osmotic conditions. Alternatively, the small
Rab11-positive vesicles observed at the cell surface of cells suspended in
hypo-osmotic environment might be involved in trafficking water out of
cells.
It is interesting that cells overexpressing DdRab11N125I protein have a
higher rate of phagocytosis than do wild-type cells. This is in direct
contrast to the situation observed in mammalian macrophages. Cox et al. have
reported that expression of a GTP binding-deficient Rab11 protein in
macrophages decreased transferrin recycling and phagocytosis, implying that in
mammalian cells Rab11 may regulate transport of membrane from recycling
endosomes to the forming phagocytic cup (Cox et al.,
2000). Expression of
DdRab11N125I resulted in the accumulation of swollen CV reticulum, suggesting
that DdRab11 may regulate the level of membrane in the reticular elements of
the CV. If the reticular elements of the CV contributed membrane to the
forming phagocytic cup, then an increase in this pool of membranes would be
predicted to increase the rate of phagocytosis. Another possibility is that a
shared effector protein exists between the contractile vacuolar and phagocytic
systems, and that the dominant negative DdRab11 protein is `soaking up' this
effector resulting in a reduction in the rate of phagocytosis.
In this study, we present evidence that DdRab11 associates almost exclusively with the CV system of membranes, including both reticular elements and bladder membranes. In addition, it appears that DdRab11 may regulate membrane traffic to or from the CV, and, along with at least one other Rab, appears to be crucial in the formation of an organelle important in osmotic regulation and perhaps recycling of internalized membrane proteins back to the cell surface.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Becker, M., Matzner, M. and Gerisch, G. (1999).
Drainin required for membrane fusion of the contractile vacuole in
Dictyostelium is the prototype of a protein family also represented
in man. EMBO J. 18,3305
-3316.
Buczynski, G., Bush, J. Zhang, L., Rodriguez-Paris, J. and Cardelli, J. (1997). Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum. Mol. Biol. Cell 8,1343 -1360.[Abstract]
Bush, J. and Cardelli, J. (1993). Molecular cloning and DNA sequence of a Dictyostelium cDNA encoding a Ran/TC4 related GTP binding protein belonging to the Ras superfamily. Nucleic Acids Res. 21,1675 .[Medline]
Bush, J., Franek, K., Daniels, J., Spiegelman, G., Weeks, G. and Cardelli, J. (1993). Cloning and characterization of three novel Dictyostelium discoideum Rab family genes and two genes related to human Rab-1. Gene 136, 55-60.[Medline]
Bush, J., Nolta, K., Rodriguez-Paris, J., Ruscetti, T.,
Temesvari, L., Steck, T. and Cardelli, J. (1994). A Rab4-like
GTPase colocalizes with V-H(+)-ATPases in extensive reticular elements of the
contractile vacuoles and lysosomes in Dictyostelium discoideum. J.
Cell Sci. 107,2801
-2812.
Bush, J. and Cardelli, J. (1995). Rab and Rho proteins in Dictyostelium discoideum. In Guidebook to the Small GTPases (ed. M. Zerial, L. Huber and J. Tooze), pp.385 -390. New York: Oxford University Press.
Bush, J., Temesvari, L., Rodriguez-Paris, J., Buczynski, G. and Cardelli, J. (1996). A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum. Mol. Biol. Cell. 7,1623 -1638.[Abstract]
Calhoun, B. and Goldenring, J. (1996). Rab proteins in gastric parietal cells: evidence for the membrane-recycling hypothesis. Yale J. Biol. Med. 69, 1-8.
Calhoun, B. C., Lapierre, L. A., Chew, C. S. and Goldenring, J.
R. (1998). DdRab11 redistributes to apical secretory
canaliculus during stimulation of gastric parietal cells. Am. J.
Physiol. 275,C163
-C170.
Cox, D., Lee, D., Dale, B., Calafat, J. and Greenberg, S.
(2000). A DdRab11-containing rapidly recycling compartment in
macrophages that promotes phagocytosis. Proc. Natl. Acad. Sci.
USA 97,680
-685.
Daniels, J., Bush, J., Cardelli, J., Spiegelman, G. and Weeks, G. (1994). Isolation of two novel Ras genes in Dictyostelium discoideum; evidence for a complex developmentally regulated Ras gene subfamily. Oncogene 9, 501-509.[Medline]
Dragoi, I. and O'Halloran T. (1998). Cloning and characterization of a Dictyostelium gene encoding a small GTPase of the Rab11 family. J. Cell Biochem. 70, 29-37.[Medline]
Gabriel, D., Hacker, U., Kohler, J., Muller-Taubenberger, A.,
Schwartz, J. Westphal, M. and Gerisch, G. (1999) The
contractile vacuole network of Dictyostelium as a distinct organelle: its
dynamics visalized by a GFP marker protein. J. Cell
Sci. 112,3995
-4005.
Green, E., Ramm, E., Riley, N., Spiro, D., Goldenring, J. and Wessling-Resnick, M. (1997). DdRab11 is associated with transferrin-containing recycling compartments in K562 cells. Biochem, Biophys. Res. Commun. 239,612 -616.[Medline]
Hall, A. (1993). Ras-related proteins. Curr. Opin. Cell Biol. 5, 265-268.[Medline]
Heuser, J., Zhu, Q. and Clarke, M. (1993). Proton pumps populate the contractile vacuoles of Dictyostelium discoideum. J. Cell Biol. 121,1311 -1327.[Abstract]
Maniak, M. (2001). Fluid-phase uptake and transit in axenic dictyostelium cells. Biochim. Biophys. Acta 1525,197 -204.[Medline]
Manstein, D., Schuster, H., Morandini, P. and Hunt, D. (1995). Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162,129 -134.[Medline]
Mellman, I. (1994). Membranes and sorting. Curr. Opin. Cell Biol. 6, 497-498.
Nolta, K. and Steck, T. (1994). Isolation and
initial characterization of the bipartite contractile vacuole complex from
Dictyostelium discoideum. J. Biol. Chem.
269,2225
-2233.
Neuhaus E. M. and Soldati T. (2000) A myosin I
is involved in membrane recycling from early endosomes. J. Cell
Biol. 150,1013
-1026.
Nuoffer, C. and Balch, W. (1994). GTPases: Multifunctional molecular switches regulating vesicular traffic. Annu. Rev. Biochem. 63,949 -990.[Medline]
Ren, M., Xu, G., Zeng, J., De Lemos-Chiarandini, C., Adesnik, M.
and Sabatini, D. (1998). Hydrolysis of GTP on Rab11 is
required for the direct delivery of transferrin from the pericentriolar
recycling compartment to the cell surface but not from sorting endosomes.
Proc. Natl. Acad. Sci. USA
95,6187
-6192.
Rodrigeuz-Paris, J., Nolta, K. and Steck, T.
(1993). Characterization of lysosomes isolated from
Dictyostelium discoideum by magnetic fractionation. J.
Biol. Chem. 268,9110
-9116.
Rupper, A. and Cardelli, J. (2001) Regulation of phagocytosis and endophagosomal trafficking pathways in Dictyostelium discoideum. Biochim. Biophys. Acta 1525,205 -216.[Medline]
Sambrooke, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sesaki, H., Wong, E. and Siu, C. (1997). The
cell adhesion molecule DdCAD-1 in Dictyostelium is targeted to the
cell surface by a nonclassical transport pathway involving contractile
vacuoles. J. Cell Biol.
138,939
-951.
Sheff, D., Daro, E., Hull, M. and Mellman, I. (1999). The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. J Cell. Biol. 5,123 -139.
Sonnichsen B., De Renzis S., Nielsen E., Rietdorf J. and Zerial
M. (2000) Distinct membrane domains on endosomes in the
recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.
J. Cell Biol. 149,901
-914.
Temesvari L., Rodriguez-Paris J., Bush J., Zhang L. and Cardelli
J. (1996). Involvement of the vacuolar proton-translocating
ATPase in multiple steps of the endo-lysosomal system and in the contractile
vacuole system of Dictyostelium discoideum. J. Cell
Sci. 109,1479
-1495.
Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. and Parton, R. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135,913 -924.[Abstract]
Urbe, S., Huber, L., Zerial, M., Tooze, S. and Parton, R. (1993). Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells. FEBS Lett. 334,175 -182.[Medline]
Zhu Q., Liu T. and Clarke M. (1993) Calmodulin
and the contractile vacuole complex in mitotic cells of Dictyostelium
discoideum. J. Cell Sci.
104,1119
-1127.