1 INSERM U528, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris
Cedex 05, France
2 CNRS UMR144, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris
Cedex 05, France
* Author for correspondence (e-mail: Corinne.Leprince{at}curie.fr)
Accepted 30 January 2003
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Summary |
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Key words: Amphiphysin, Sorting nexin, Endocytosis, Trafficking, Endosome
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Introduction |
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Another crucial element of the endocytosis machinery, dynamin, should be
added to the list of amphiphysin-interacting molecules. In this latter case,
the binding is dependent on the SH3 domain of amphiphysins and a proline-rich
region of dynamin (David et al.,
1996; Grabs et al.,
1997
; Owen et al.,
1998
). Even though the biochemical basis of dynamin function needs
to be clarified, dynamin is essential for the membrane fission reaction
leading to the formation of an individualized endocytic vesicle. In agreement
with this model, microinjection of the SH3 domain of amphiphysin 1 into the
lamprey synapse (Shupliakov et al.,
1997
) or transient transfection of the SH3 domain of amphiphysin 1
(Wigge et al., 1997
) or
amphiphysin 2 (Owen et al.,
1998
) into fibroblasts inhibited clathrin-dependent endocytosis of
membrane receptors by sequestration of dynamin. The same SH3 domain is
responsible for the binding of amphiphysin 2 to the proline-rich domain of
synaptojanins (synaptojanin 1, 2B1 and 2B2)
(Nemoto et al., 2001
), which
are phosphoinositide phosphatases crucial for clathrin uncoating of the
vesicles. Thus, amphiphysins provide a link between the clathrin-coated pits
that precede clathrin-dependent endocytosis and the newly formed endocytic
vesicle, by influencing either directly or indirectly, membrane curvature,
membrane fission and/or vesicle uncoating.
Following endocytosis, membrane proteins and their ligands are transported to early endosomes and sorted in three possible directions: directly recycling to the cell surface, transported to perinuclear recycling endosomes, or transported to lysosomes via late endosomes (Kirchausen, 2000). The sorting and transport processes within the endosomal compartment are highly regulated and rely on a molecular machinery that still needs to be fully defined.
Sorting nexins (SNX) are a family of proteins present in a number of
organisms from human to Caenorhabditis elegans and yeast. Human SNX1
was first characterized as an epidermal growth factor (EGF) receptor
interacting molecule in a two-hybrid screen with a cytoplasmic portion of the
receptor (Kurten et al.,
1996). Later reports characterized other SNX family members by
homology with SNX1, and showed that they interact with different kinds of
membrane receptors: tyrosine kinase receptors, serine-threonine kinase
receptors of the TGFß family, transferrin receptor and leptin receptor
(Haft et al., 1998
;
Parks et al., 2001
). The
binding capacities for these different kinds of receptors vary from one SNX to
another. Presently, the SNX family is still expanding (more than 20 members in
humans), essentially through searches in sequence databases.
A number of SNX molecules have coiled-coil domains in the C-terminal part
of the molecule but the most obvious structural signature is the presence of a
`Phox homology' (PX) domain, which was initially defined in the
P47phox and p40phox subunits of NADPH oxidase (Prehoda
et al., 2001). PX domains were recently shown to bind phosphoinositides, with
different PX domains having different phosphoinositide specificities. Point
mutations in the PX domain that affect phosphoinositide binding also affect
membrane attachment of SNX (Prehoda and
Lim, 2001; Xu et al.,
2001
; Kanai et al.,
2001
). Thus, PX domains join the family of
phosphoinositide-binding modules, including PH domains, FYVE domains and ENTH
domains, that participate in membrane anchorage. In addition, it remains
possible that PX domains have a general low affinity SH3-binding capacity by a
conserved poly-proline motif (Hiroaki et
al., 2001
).
The function of SNX molecules is more documented in yeast cells than in
mammalian cells. The SNX1 yeast orthologue, Vps5p, is essential for the
correct targeting of carboxypeptidase Y from the trans-Golgi network (TGN) to
a pre-vacuolar/endosomal compartment. Vps5p is a component of a multimeric
complex containing other subunits Vps26p, Vps29p, Vps35p and Vps17p.
This complex called `retromer' is believed to act as a membrane coat in which
SNX molecules can either recruit cargo proteins or participate in the
formation of the vesicle (Horazdovsky et
al., 1997). It remains to be established if similar roles can be
fulfilled by mammalian SNX molecules (Haft
et al., 2000
).
In this work, we present data showing that amphiphysin 2 and SNX4 interact, both in a yeast two-hybrid assay and in vivo in mammalian cells where they associate in the cell cytosol and on cytoplasmic vesicular structures. Overexpression of SNX4 inhibits the endocytosis of the transferrin receptor as efficiently as the SH3 domain of amphiphysin 2. At lower levels of expression, SNX4 colocalizes with transferring-containing vesicles, some of which are also amphiphysin 2 positive. Even though it is possible that SNX4 plays a role at the endocytosis step, we propose that the amphiphysin 2/SNX4 partnership is important for the control of the endosome fate, after the formation of the endocytic vesicle.
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Materials and methods |
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Antibodies
The antibodies used in this study were a mouse monoclonal anti-myc (clone
9E10; Santa Cruz, CA and Roche Meylan, France), a rabbit polyclonal anti-myc
antibody (Upstate Biotechnology), mouse monoclonal anti-EEA1, anti-CD63,
anti-LAMP1 and anti-calnexin antibodies (all from Becton Dickinson
Transduction Laboratories, San Diego, CA), and a mouse monoclonal anti-BIN1
antibody (Upstate Biotechnology, Lake Placid, NY). The rabbit polyclonal
anti-Rab11 antibody was a kind gift from J. Salamero (CNRS UMR144, Institut
Curie, Paris). A previously generated rabbit polyclonal antiserum against a
C-terminal part of murine Bramp2 (Leprince
et al., 1997) was proven to be mouse-specific.
cDNA constructs
Different portions of mouse amphiphysin 2 cDNA were cloned by PCR in yeast
expression vectors. Briefly, full length Amp2m previously described in
macrophages (Gold et al., 2000)
or partial cDNAs corresponding to residues 1-146, 147-410 and 1-304 of Amp2m
were cloned in the pLex10 vector producing a N-terminal fusion protein with
the DNA-binding domain of LexA. The full sequence of mouse Amp2m is registered
in GenBank under the accession number AF068915.
Full length human SNX4 cDNA (Kurten et
al., 1996) and partial cDNAs corresponding to residues 1-367 and
1-404 of SNX4 were cloned in the pGAD1318 vector, producing a N-terminal
fusion protein with the activation domain of GAL4.
Full length cDNAs of mouse Amp2m, mouse Bramp2
(Leprince et al., 1997), human
SNX4 and a partial cDNA corresponding to residues 368-450 of SNX4 were cloned
by PCR in different vectors containing a CMV promoter for mammalian cell
expression, pRK5 or pRK5myc. All constructs were verified by DNA
sequencing.
Rab5 cDNA and Rab11 cDNA cloned in a GFP-fusion expression vector were kind
gifts from P. Chavrier (CNRS UMR144, Institut Curie, Paris) and RGS14 cDNA
cloned in pRK5myc was kindly given by S. Traver
(Traver et al., 2000).
Two-hybrid screening
Two-hybrid screening was performed in the L40 yeast strain. An N-terminal
portion of murine amphiphysin 2 (residues 1-304) expressed in fusion with the
LexA DNA-binding domain was used as a bait to screen a Jurkat cell two-hybrid
cDNA library (Leprince et al.,
1997; Vojtek et al.,
1993
). Yeast cells co-transformed with pLex and pGAD vectors are
able to grow on leucine-and tryptophan-deficient media. Yeast clones producing
proteins that interact with amphiphysin 2 were selected on media deficient in
leucine, tryptophan and histidine. Interaction between the bait and a
potential partner was confirmed in a beta-galactosidase assay since LacZ
expression in L40 is dependent on the reconstitution of a functional LexA/GAL4
transcription factor. For each selected yeast clone, the pGAD plasmid was
purified, reamplified in DH5alpha bacteria and the insert was sequenced.
Two-hybrid assays were also used for testing selected protein-protein interactions with full length or partial mouse amphiphysin 2 and human SNX cloned in pLex and pGAD vectors, respectively.
Cell line transient transfections
3T3 cells were transiently transfected with SNX4 in pRK5myc using
Lipofectamine Plus according to the manufacturer's instructions (Life
Technologies). For biochemistry experiments, cells were plated at
2x106 per dish one day before transfection, transfected with
0.4 µg of DNA and cultured for an additional 24 hours. For
immunofluorescence analysis, cells were plated on coverslips at
6x104 per well in a 24 well-plate, transfected one day later
and cultured for an additional 24 hours.
HeLa cells were transiently transfected by using either the calcium phosphate method for immunoprecipitations experiments or Exgen 500 (Upstate Biotechnology) for immunofluorescence. Briefly, for subsequent immunoprecipitation experiments, cells were plated at 1x106 per dish in fresh medium. One day later, cells were added with a mix of 5-10 µg DNA in calcium-containing buffer, cultured for 16 hours, washed with fresh medium and cultured for an additional 24 hours before lyses and biochemical experiments. For immunofluorescence analysis, cells were plated on coverslips at 3x104 per well in a 24 well-plate. DNA was mixed with Exgen 500 (Upstate Biotechnology) according to the manufacturer's instructions and added to cells. HeLa cells were cultured for additional 16-24 hours before immunofluorescence assay.
Immunoprecipitations and western blotting assays
Transfected cells were washed in cold PBS and resuspended by scrapping in
cold hypotonic buffer (10 mM HEPES pH 7.5, 10 mM NaCl, 1 mM EDTA) containing a
protease inhibitor cocktail (Roche) supplemented with 1 µg/ml pepstatin, 1
mM Na3VO4 and 50 mM NaF (all from Sigma-Aldrich). Cells
were disrupted in a dounce homogeneiser and cell extracts were centrifuged at
1500 g to remove nuclei, intact cells and debris. The
resulting supernatants (PNS: post nuclear supernatant) were ultracentrifuged
for 30 minutes at 100,000 g. The pellets containing cell
membranes were washed once in hypotonic buffer and resuspended in 10 mM Tris
pH 7.5, 150 mM NaCl, and 1% NP40 (all from Sigma-Aldrich). The membranes were
resuspended by pipetting, and rotated for 30 minutes at 4°C in order to
achieve maximum solubilization. The unsolubilized material was removed by
centrifugation at 10,000 g for 30 minutes. The supernatants of
the 100,000 g ultracentrifugations (cytosolic fractions) were
supplemented with Tris, NaCl and NP40 in order to reach the concentrations
mentioned above.
The membrane and cytosolic fractions were submitted to immunoprecipitations by incubation with anti-myc or anti-amphiphysin 2 antibody for 2 hours at 4°C. Immune complexes were recovered by using Protein A-Sepharose beads (Roche) and washed 3 times in 10 mM Tris pH 7.5, 150 mM NaCl, 1% NP40. Immunoprecipitation products were run on SDS-PAGE and transferred to nitrocellulose (Hybond ECL, Amersham-Pharmacia, Orsay, France). After saturation in 10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20 containing 5% non-fat powdered milk, membranes were blotted with relevant antibodies, followed by a secondary HRPO-conjugated antibody. Immunoreactive bands were revealed by ECL (Amersham-Pharmacia).
Immunofluorescence analysis
HeLa or 3T3 cells were transiently transfected with various cDNAs. After 24
hours of transfection, cells were washed in cold PBS and fixed in 4%
paraformaldehyde (Sigma-Aldrich) for 30 minutes at 4°C, unless they are
pre-permeabilized. In this latter case, cells were incubated for 5 minutes at
4°C in 80 mM PIPES pH 6.8, 5 mM EGTA, 1 mM MgCl2 containing
0.1% bovine serum albumin (BSA fraction V, Sigma-Aldrich) and 0.01% saponin
(Sigma-Aldrich). After 3 washes in cold PBS, cells were fixed in 4%
paraformaldehyde for 30 minutes.
Thereafter, pre-permeabilized or not, cells were washed three times in cold PBS, incubated for 15 minutes in PBS containing 50 mM NH4Cl, washed again and incubated for 10 minutes with 0.1% saponin. Incubation with primary antibodies diluted in immunofluorescence buffer (IF-buffer: PBS containing 1% BSA and 0.1% saponin) was performed for 30 minutes. After three washes in IF-buffer, cells were labeled for 30 minutes with secondary antibodies coupled to the relevant fluorochrome (Alexa 488-coupled antibodies from Molecular Probes, or Cy-3- and Cy-5-coupled antibodies from Jackson Laboratories). Coverslips were washed in IF buffer, then in PBS and mounted in Mowiol (Sigma-Aldrich).
Cell images were acquired by confocal microscopy with a Leica SP2. Digital monochrome images were collected for each appropriate channel and pseudo-colored with Metamorph (Universal Imaging).
Endocytosis assays
For transferrin receptor endocytosis assay, cells were preincubated for 1
hour in serum-free DMEM containing 20 mM HEPES pH 7.5 at 37°C. Endocytosis
of Alexa 488-conjugated transferrin (Molecular Probes) was performed at
37°C for 15 minutes in endocytosis medium (DMEM, 20 mM Hepes pH 7.5, 1
mg/ml BSA) containing 50 µg/ml Alexa 488-conjugated transferrin. Cells were
rapidly cooled at 4°C, washed twice in cold PBS, and fixed with 4%
paraformaldehyde for 3 hours at 4°C. Then, cells were processed for
indirect immunofluorescence as described above.
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Results |
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Among all the interacting clones selected in the two-hybrid screen, we identified several partial cDNAs coding for the same protein, sorting nexin 4 (SNX4). Three of them (residues 408-450, 377-450 and 292-450) are presented in Fig. 1. They code for C-terminal portions of SNX4, which also interacted with full-length amphiphysin 2.
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SNX4 was initially characterized as a SNX1 homologue able to interact with
membrane receptors such as EGF, PDGF, insulin or leptin receptors
(Haft et al., 1998). As with
many other members of the SNX family, SNX4 presents a N-terminal Phox homology
(PX) domain (Prehoda and Lim,
2001
) and a C-terminal coiled-coil region. The shortest clone
isolated in the screen (residues 408-450) defined a C-terminal 42 amino-acid
region, just after the SNX4 coiled-coil domain, as the domain interacting with
amphiphysin 2. Longer C-terminal parts of SNX4 (residues 377-450 and 292-450)
including the coiled-coil domain kept their binding capacities. However,
N-terminal parts of SNX4 (residues 1-367 and 1-404) and full length SNX4 were
deficient in interacting with amphiphysin 2. Thus, full length SNX4
even though well expressed in yeast (data not shown) has a general
conformation that does not allow interaction with amphiphysin 2 in the
conditions of the two-hybrid assay.
In order to define the region of amphiphysin 2 involved in the interaction, we cloned different portions of amphiphysin 2 in yeast expression vectors. As shown in Fig. 2B, the partial amphiphysin 2 construct (residues 147-410) codes for a polypeptide that interacted with C-terminal SNX4, as well as the partial amphiphysin 2 used in the screen (residues 1-304). This suggests that the internal region of amphiphysin 2 (Amp2m), starting at the second coiled-coil (residue 147) and ending at residue 304, is important for the interaction with SNX4. The absence of interaction obtained with the N-terminal part (residues 1-146) suggests that this part of the molecule is not necessary for binding to SNX4. Because the longer isoform of amphiphysin 2 (Bramp2/Amp2a highly expressed in brain) gave a high background in two-hybrid assays (data not shown), the interaction of SNX4 with Bramp2 was not addressed by this method.
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SNX4 and amphiphysin 2 can be co-immunoprecipitated in vivo
The interaction between amphiphysin 2 and SNX4 was confirmed by using a
GST-amphiphysin 2 (Amp2m) and in vitro 35S-labeled SNX4 (residues
368-450) (data not shown).
In order to assess whether this interaction existed also in mammalian
cells, immunoprecipitations were performed first with murine 3T3 cells
(Fig. 2A) and second with human
HeLa cells (Fig. 2B). Good
anti-SNX4 antibodies were not available for such experiments, inciting us to
overexpress myc-SNX4 and a control myc-RGS14
(Traver et al., 2000) by cell
transfection. In membrane (M) and cytosol (C) total extracts, myc-SNX4 and
myc-RGS14 were detected at 55-60 kDa and 65-70 kDa, respectively
(Fig. 2A, left panel). A mouse
amphiphysin 2 specific antibody was able to immunoprecipitate the endogenous
murine amphiphysin 2 as a 60-70 kDa doublet
(Fig. 2A, right panel). The
immunoprecipitated amphiphysin 2 was associated with myc-SNX4 in the cytosol
fraction and to a lesser extent in the membrane fraction. Control myc-RGS14
was not immunoprecipitated (Fig.
2A, central panel).
Such physical association between amphiphysin 2 and SNX4 was confirmed in HeLa cells that have been transfected with Bramp2/Amp2a and myc-SNX4 (Fig. 2B). Membrane and cytosol total extracts contained the 55-60 kDa myc-SNX4, the 65-70 kDa myc-RGS14 and a 80-90 kDa doublet of Bramp2. Overexpressed amphiphysin 2/Bramp2 was easily detected in the immunoprecipitations of myc-SNX4 from both cytosolic and membrane fractions, but not in the immunoprecipitations of the control myc-RGS14.
Taken together, these results indicate that the endogenous as well as the overexpressed amphiphysin 2 can indeed interact with full length SNX4 in the context of mammalian cells.
Intracellular localization of SNX4 and amphiphysin 2
To acquire further insight into the capacity of SNX4 and amphiphysin 2 to
meet and to cooperate in entire cells, we compared their respective
subcellular localizations. HeLa cells were transiently transfected with
myc-amphiphysin 2 (mouse Amp2m or Bramp2) or myc-SNX4 and then labeled with
anti-myc antibody. As shown in Fig.
3A,B transfected amphiphysin 2 was highly expressed throughout the
cell and exhibited an important cytosolic staining. The same images were
obtained with cells transfected with non-tagged amphiphysin 2 revealed with a
specific anti-BIN1 antibody or our polyclonal rabbit anti-Bramp2 (data not
shown). Both antibodies were unable to detect endogenous amphiphysin 2 in HeLa
cells, the latter one because of its mouse specificity. SNX4 showed a more
discrete localization characterized by a major punctate distribution, probably
on vesicular structures, and a minor cytosolic diffuse staining
(Fig. 3C). In conditions of
pre-permeabilization with 0.01% saponin, which removes a great part of the
cytosol, amphiphysin 2 (Amp2m and Bramp2) also presented a punctate
distribution, reflecting the membrane-associated localization of the remaining
amphiphysin 2 (Fig. 3D,E).
These latter images were similar to the images obtained with myc-SNX4
transfected cells (Fig. 3F)
even though SNX4 staining displayed bigger patches of fluorescence. When both
labeling images were analyzed in parallel, in the same pre-permeabilized and
co-transfected HeLa cells, it was evident that myc-SNX4 and amphiphysin 2
(Amp2m or Bramp2) colocalized to a great extent
(Fig. 3G-O). The same
conclusion was drawn from 3T3 cells in which our polyclonal anti-amphiphysin 2
antibody was able to recognize the endogenous amphiphysin 2 (at least the 60
and 70 kDa isoforms seen in immunoprecipitations). As for HeLa cells,
amphiphysin 2 was clearly distributed throughout the cytoplasm and
pre-permeabilization with 0.01% saponin helped to visualize a punctate
staining due to a vesicular amphiphysin 2. In 3T3 cells, transfected myc-SNX4
colocalized at least in part with endogenous amphiphysin 2
(Fig. 3P-R).
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In a second step, we tried to characterize the subcellular structures
containing the SNX4 staining on one hand and the amphiphysin 2 staining on the
other hand. Myc-SNX4 transfected HeLa cells were labeled with anti-myc
antibody in parallel with specific antibodies for the endogenous markers EEA1,
Rab11, CD63, LAMP1, calnexin, or in parallel with GFP-Rab5. As shown in
Fig. 4, myc-SNX4 colocalized
with the early endosomal markers, EEA1 and Rab5. CD63, a marker for late
endosomes/lysosomes, and even less Rab 11, a marker for recycling endosomes,
were marginally present on SNX4 positive structures. Other markers such as
LAMP1 for lysosomes, calnexin for the endoplasmic reticulum or a Golgi
specific marker (data not shown) did not colocalize with SNX4. Thus, SNX4 can
be added to the growing list of SNX family members presenting an early
endosomal localization (Teasdale et al.,
2001; Kurten et al.,
2001
). Yet this localization does not account for all of the SNX4
positive vesicular structures, as was the case for SNX1
(Zhong et al., 2002
).
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In Fig. 5, amphiphysin 2 distribution was analyzed after transfection of myc-Bramp2/Amp2a in parallel with the same endogenous markers as above: EEA1, CD63, LAMP1 were detected by antibodies and Rab11 was detected by GFP-Rab11. Bramp2 gave a reticulo-vesicular staining pattern, which was less clustered than SNX4, and partially colocalized with EEA1, CD63, LAMP1 and calnexin. This suggests that amphiphysin 2, which is largely distributed throughout the cell body, can be associated with different types of endosomal structures, from early to late endosomes/lysosomes and with endoplasmic reticulum. Amphiphysin 2/SNX4 interaction may take place on part of these vesicles, for example on early endosomes as could be seen in a three-color analysis with anti-EEA1 antibody (data not shown).
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Inhibition of transferrin receptor endocytosis after SNX4
overexpression
Amphiphysin 2 helps to recruit key elements of the endocytosis machinery
such as dynamin. Previous studies have showed that overexpression of the SH3
domain of amphiphysins inhibits the early steps of endocytosis by
sequestration of dynamin. We tested whether partial or full length SNX4 was
able to interfere with endocytosis. As expected, the SH3 domain of amphiphysin
2 was a very potent inhibitor of transferrin receptor endocytosis
(Fig. 6), whereas an internal
domain of amphiphysin 2 (residues 47-142 between the two coiled-coil regions)
had no effect. In the same experimental conditions, full length SNX4 and a
C-terminal part of SNX4 (residues 368-450) both tagged with a myc
epitope were also very efficient in inhibiting transferrin receptor
endocytosis (Fig. 6, lower
panels). Such an interference of SNX4 with the endocytic process could be due
to a vesicular trafficking block but no particular accumulation of transferrin
could be seen in a juxta-plasma membrane vesicular compartment. Another
possibility is that SNX4 might have functional relationships with key elements
of the endocytic machinery, acting itself on endocytosis or by sequestration
of key elements of endocytosis, in the wrong subcellular location and/or in a
non-functional state. One of such elements is likely to be amphiphysin 2.
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In conditions of lower expression, myc-SNX4 was no longer inhibitory for transferrin receptor endocytosis. Fig. 7A (upper panels) gives a good representation of three different myc-SNX4 expression levels (high, intermediate and very low/null) giving no, intermediate or very good transferrin receptor endocytosis, respectively. The lower panels (Fig. 7A) are magnifications of an intermediate state. Transferrin containing vesicles are clearly SNX4 positive. A fraction of them are early endosomes labeled with EEA1 (data not shown). But most importantly, a fraction of these transferrin-containing vesicles are double positive for SNX4 and amphiphysin 2 as can be shown in a three-color experiment (Fig. 7B, white dots). This image is an instantaneous representation of a dynamic process in which transferring-containing vesicles that are recycling back to the plasma membrane are the site of interaction between amphiphysin 2 and SNX4. Taken together, these results suggest that amphiphysin 2 is playing a role from endocytosis to endosomal trafficking. One of its regulatory roles may involve its partner SNX4.
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Discussion |
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The association between amphiphysin 2 and SNX4 has been identified in a yeast two-hybrid screen with the N-terminal half of amphiphysin 2 (Amp2m, residues 1-304) and a Jurkat oligodT cDNA library. The minimal region of interaction that we were able to define for SNX4 is a C-terminal 42 amino acid region, just after the coiled-coil domain. For amphiphysin 2, a central portion starting at the second coiled-coil domain and ending at residue 304 seems to be required.
SNX4 is the only member of the SNX family that we isolated in our two-hybrid screen, raising the question of the specificity of its interaction with amphiphysin 2. In preliminary experiments, C-terminal parts of human SNX1 (including or not 1, 2 or 3 coiled-coil domains) were unable to bind amphiphysin 2 in two-hybrid assays (data not shown). Even though a more extensive interaction study would be necessary with the numerous members of the SNX family, this result is consistent with the very poor amino acid similarity between the SNX4 C-terminal region (42 residues) and other SNX sequences. In any case, as SNX molecules have the ability to form homo- and hetero-oligomers, amphiphysin 2 might associate with different SNX even indirectly within such an oligomeric complex. The question of interaction specificity is also important for amphiphysin 2, which is expressed as multiple molecular isoforms. Co-immunoprecipitation experiments showed that SNX4 associates with the long Bramp2/Amp2a overexpressed in HeLa cells, and also with shorter isoforms naturally expressed in 3T3 cells as a 60-70 kDa doublet.
In 3T3 and HeLa cells, myc-tagged SNX4 was present primarily on
reticulo-vesicular structures and secondarily in the cytosol. Part of the SNX4
positive vesicles are early endosomes, in agreement with previous works
showing that the intracellular distributions of a number of SNX molecules show
colocalization with early endosomal markers
(Teasdale et al., 2001;
Barr et al., 2000
). But SNX4 is
distributed beyond the early endosomal compartment, on a greater population of
vesicles, a characteristic that was also pointed out for SNX1
(Zhong et al., 2002
).
In the same two cell types, amphiphysin 2 was highly expressed in the
cytosol and to a lesser extent in association with membranes, as documented by
biochemical and immunofluorescence analysis. The same kinds of images were
obtained with HeLa cells expressing moderate levels of myc-tagged amphiphysin
2 and 3T3 cells expressing a native amphiphysin 2. In an immunofluorescence
assay including a pre-permeabilization step, amphiphysin 2 exhibited a
punctate staining on reticulo-vesicular structures and a partial
colocalization with early to late endosomes/lysosomes and with endoplasmic
reticulum. The endosomal localization of amphiphysin 2 is consistent with
previous studies demonstrating that amphiphysin 2 could associate with early
phagosomes in macrophages (Gold et al.,
2000). The limited aspect of the co-staining between amphiphysin 2
and organelle markers is due to their very different distributions: throughout
the cell body for amphiphysin 2 and in restricted cell compartments for the
organelle markers.
The presence of amphiphysin 2 on vesicles that are far from the plasma membrane and are far from being strict endocytic vesicles, combined with its colocalization with SNX4 suspected to regulate intracellular trafficking, suggests that amphiphysin 2 may have a post-endocytic role on the endocytic vesicle and on the following endosome.
Besides being an adaptor for components of the endocytosis machinery that
assemble at the neck of the nascent endocytic vesicle, amphiphysin 2 has been
previously implicated in a number of other processes such as tumor progression
or cytoskeleton organization. BIN1, one of the amphiphysin 2 isoforms with a
nuclear localization signal, was shown to interact with myc and suppress its
tumor promoting effect (Sakamuro et al.,
1996). Other studies in different cell types have documented an
interaction of amphiphysin with the actin cytoskeleton. First, mutations in
RVS167 or RVS161 genes two yeast homologs of
amphiphysin produced defects in the peripheral actin cytoskeleton,
whereas normal Rvs167p localized to actin rich cortical patches
(Sivadon et al., 1995
;
Balguerie et al., 1999
).
Second, in muscle cells, the shortest amphiphysin 2 isoforms were shown to be
highly expressed (Butler et al.,
1997
) and localize around the submembranous cytoskeleton of
T-tubules. These data were emphasized by recent studies in Drosophila
where a unique gene, amph, is responsible for the expression of
amphiphysin. Mutant flies do not show any particular deficiency in the
endocytosis of synaptic vesicles but they have a muscle cell defect with
reduced and mislocalized T-tubule projections
(Razzaq et al., 2001
;
Zelhof et al., 2001
;
Leventis et al., 2001
). In
mammalian cells, an amphiphysin 2 isoform displaying an additional short
sequence (encoded by exon 10) was also shown to induce tubular membrane
invaginations, particularly critical for muscle cell morphology
(Lee et al., 2002
). Looking at
an extended series of cell types, drosophila studies further suggest that
amphiphysin is always present in membrane domains that undergo great changes
in curvature and surface area, for example in the apical membrane of
epithelial cells. Thus, amphiphysin seems to be essential for a series of
membrane movements and this biological effect may rely on its connection with
the actin cytoskeleton, its ability to tubulate lipids and/or its docking
potential for a number of proteins (Zhang et al., 2002).
This new aspect of amphiphysin function reinforces the importance of the interaction that we describe herein between amphiphysin 2 and SNX4, a molecule that is suspected to play a role in vesicular trafficking. If amphiphysin is able to regulate membrane morphology and movements, it is conceivable that the amphiphysin 2 present around an endosome could regulate the budding events that are crucial for intracellular trafficking. Its partnership with SNX4 can be seen as another level of regulation with the same goal of vesicular trafficking. SNX molecules have been shown to associate with membrane receptors such as EGF, PDGF, insulin, transferring and leptin receptors. In this regard, the interference of SNX4 with transferrin receptor endocytosis that we documented herein may be due to a direct effect of SNX4 on the endocytic process. But another possibility is that the endocytosis inhibition is just a sequestration of key elements of endocytosis. One of these elements may be amphiphysin 2 and the inhibition is rendered possible by amphiphysin 2 pleiotropic functions, playing a role in endocytosis, as well as vesicular trafficking. Inside a vesicular coat, SNX4 is suspected to act either on the selection of cargo proteins or on the membrane budding/fusion processes. We documented above the simultaneous presence of SNX4 and amphiphysin 2 on transferring-containing vesicles that are on their way for recycling. This is an instantaneous picture of a dynamic process that involves not less dynamic molecular interactions. At the surface of the endocytic vesicle or of the endosome, it is conceivable that the interaction between SNX4 and amphiphysin modifies SNX4 conformation and, consequently, either its membrane anchorage, its interaction with other components of the coat, or its functional effect on membrane movements that still need to be well defined. Reciprocally, the same molecular interaction may modify amphiphysin ability to act on membranes dynamics, i.e. its connection with the actin cytoskeleton, its ability to tubulate lipids or its adaptor potential in the aim to regulate vesicular trafficking. Further studies will be necessary to define the exact contribution of amphiphysin 2 and SNX4 taken individually or in association in the molecular machinery underlying vesicular trafficking.
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