Department of Biochemistry and Biophysics, University of California San Francisco, Genentech Hall, 600 16th Street, San Francisco, California, 94143-2140, USA
* Author for correspondence (e-mail: rkelly{at}research.ucsf.edu)
Accepted 25 November 2002
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Summary |
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Key words: Synaptotagmin VII, Clathrin-dependent endocytosis, Clathrin-independent endocytosis, Sorting signals
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Introduction |
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The 13 current members of the synaptotagmin family of proteins share a
unique domain configuration: a short N-terminal region followed by a
transmembrane sequence and a long cytoplasmic tail
(Perin et al., 1991;
Sudhof, 2002
) consisting of
two tandem C2 domains (the C2A domain and C2B domain) that were originally
identified as calcium-binding domains in protein kinase C
(Kikkawa et al., 1989
).
Synaptotagmin I, a neuronal isoform present in synaptic vesicles and secretory
granules (Matthew et al.,
1981
), is the best-studied member of this family. Its role in
exocytosis was first suggested by biochemical studies that showed that the C2A
domain is responsible for the calcium-dependent association of synaptotagmin I
with acidic phospholipids and with syntaxin
(Sudhof and Rizo, 1996
),
although recent in vivo studies have suggested that these particular
interactions are not the sole means by which synaptotagmin promotes membrane
fusion (Fernandez-Chacon et al.,
2001
; Robinson et al.,
2002
). The calcium-dependent interactions of the C2B with
phospholipids, or with itself, however, may play a critical role in
neurotransmitter release in vivo (Mackler
et al., 2002
). Additionally, the C2B also participates in a number
of calcium-independent interactions with inositol polyphosphates
(Fukuda et al., 1994
), with
the t-SNARE SNAP-25 (Gerona et al.,
2000
; Schiavo et al.,
1997
) and with the `synprint region' of N- and P/Q-type
Ca2+ channels (Kim and
Catterall, 1997
; Sheng et al.,
1997
). Furthermore, mice homozygous for a mutation in the
synaptotagmin I gene have a severe defect in the fast synchronous,
calcium-dependent exocytosis of neurotransmitter
(Fernandez-Chacon et al.,
2001
; Geppert et al.,
1994
), which is consistent with a role for synaptotagmin I in
mediating calcium dependence of synaptic vesicle exocytosis. Similarly, in
Drosophila mutants, evoked release and calcium-dependent release were
severely depressed (Adolfsen and Littleton,
2001
). However, synaptotagmin I does not seem to be required for
secretory granule exocytosis in neuroendocrine cells
(Shoji-Kasai et al., 1992
),
which suggests that there are molecular differences between synaptic vesicle
and secretory granule fusion with the plasma membrane.
Synaptotagmin VII is a close relative to synaptotagmin I and is nearly as
abundant as synaptotagmin I. In contrast, however, expression of synaptotagmin
VII is not restricted to neurons; rather it is found ubiquitously in many
tissue types (Ullrich and Sudhof,
1995). Moreover, synaptotagmin VII is localized to secretory
lysosomes in epithelial cells and fibroblasts
(Caler et al., 2001
;
Martinez et al., 2000
). The
secretion of these synaptotagmin-VII-containing lysosomes was found to be
Ca2+ regulated (Martinez et
al., 2000
), mirroring the regulated secretion of
synaptotagmin-I-containing synaptic vesicles
(Andrews, 2000
;
Gerasimenko et al., 2001
). In
addition, synaptotagmin VII plays a role in dense core vesicle exocytosis in
PC12 cells (Shin et al., 2002
;
Sugita et al., 2001
) and in
insulin-containing secretory granule exocytosis in pancreatic ß-cells
(Gao et al., 2000
;
Gut et al., 2001
).
Synaptotagmin VII, therefore, contributes to secretory granule and secretory
lysosome exocytosis just as synaptotagmin I contributes to synaptic vesicle
exocytosis.
The connection between synaptotagmin and endocytosis has only recently
begun to be understood. When synaptotagmin I was disrupted in C.
elegans, a marked depletion of synaptic vesicles was seen at nerve
terminals (Jorgensen et al.,
1995), which implies a role for synaptotagmin I in vesicular
recycling. Moreover, synaptotagmin I contains a conserved high-affinity
binding site for AP-2 (Zhang et al.,
1994
). Blocking action of this site by overexpression of the
synprint region of N- and P/Q-type Ca2+ channels had an inhibitory
effect on transferrin receptor endocytosis in non-neuronal cells
(Haucke et al., 2000
). In
addition, overexpression of a synaptotagmin VII domain for oligomerization
inhibits LDL uptake and clathrincoated pit formation
(von Poser et al., 2000
). This
group of studies implicates synaptotagmins in several forms of endocytosis.
Moreover, the interaction of synaptotagmin I with AP-2 is strengthened in the
presence of tyrosine-containing domains needed for cargo internalization
(Haucke and De Camilli, 1999
);
an attractive model is that synaptotagmins help recruit clathrin cages in
areas of high cargo concentration. They could themselves be cargo for
endocytosis or they could remain at the cell surface, passively facilitating
coat recruitment.
Synaptotagmin I is itself internalized. Surprisingly, the internalization
signal of synaptotagmin I is not identical to and does not require the
AP-2-binding site. The internalization signal was found to be in a region of
the C2B domain near the C-terminus
(Blagoveshchenskaya et al.,
1999; Jarousse and Kelly,
2001a
). The region of the C-terminus critical for endocytosis was
the WHXL motif (N. Jarousse, J. Wilson, D. Arac, J. Rizo and R.B.K.,
unpublished). Regulation of this internalization signal was shown to be
responsible for tissue-specific endocytosis of synaptotagmin I
(Jarousse and Kelly, 2001a
).
Here we show that synaptotagmin VII is not actively internalized in neuronal,
fibroblast and epithelial cell types despite having the AP-2-binding site and
the WHXL motif, which are important for internalization of synaptotagmin I.
Because the internalization signals of synaptotagmin I are latent in some cell
types owing to inhibitory elements within the cytoplasmic domain, we looked
for internalization signals and inhibitory interactions within the tail of
synaptotagmin VII. Out of context, the C-terminal tail (CT) of synaptotagmin
VII's C2B was highly endocytosed in a WHXL-dependent manner, identically to
the homologous section of synaptotagmin I. In contrast to synaptotagmin I,
synaptotagmin VII has a second internalization signal in its C2A domain that
lacks both the AP-2-binding site and the previously identified C-terminal
WHXL. The homologous WKXL motif in the C2A appeared to play no role in the
domain's internalization properties. Although the CT is internalized in a
dynamin- and eps15-dependent manner, the C2A takes an unconventional pathway
that is independent of both of these proteins. The WHXL-based internalization
motif in synaptotagmin VII is normally latent since the C2B does not
internalize. The availability of two C2B domains with the same internalization
signal but different internalization properties allowed us to map out the
region that confers latency in the case of synaptotagmin VII. It was found to
reside in the 37 amino acids corresponding to the first two ß-strands of
synaptotagmin VII's C2B domain. This subdomain was transplantable and conceals
or regulates the endocytic signals in the context of either C2B or the
full-length synaptotagmin VII protein. Here we have identified two strong
endocytic signals in synaptotagmin VII that are concealed by inhibitory
elements in the C2B domain. These data suggest that synaptotagmin VII normally
acts as a passive facilitator of endocytosis, remaining on the cell surface
until special circumstances, as yet unknown, reveal its latent internalization
signals.
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Materials and Methods |
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Reagents and antibodies
The pEGFP plasmid encoding rat synaptotagmin VIIs was provided by Norma
Andrews (Yale University, New Haven, CT). The pBMN-Z-I-Neo plasmid was
provided by Don Ganem (University of California at San Francisco, San
Francisco, CA).
The monoclonal antibody against the lumenal domain of the human CD4 (clone Q4120) was obtained from the Medical Research Council AIDS Reagents Program (National Institute for Biological Standards and Control) for use in internalization assays. A second monoclonal antibody against the lumenal domain of CD4 (Pharmigen, clone RPA-T4) was used for immunofluorescence and flow cytometry.
Constructs
For the CD4-synaptotagmin constructs, a CD4 fragment (corresponding to
residues 1-426) encoding the lumenal, transmembrane and 12 amino acids of the
cytoplasmic region of the human CD4 was amplified by PCR from pBMN-Syt 1
(Jarousse and Kelly, 2001a).
The primers were chosen so that the CD4 coding region was downstream of a
BamHI restriction site and upstream of a BstBI restriction
site followed by a stop codon and SalI restriction site. This
fragment was digested with BamHI and SalI and inserted into
the corresponding sites in pBMN-Z-I-Neo to generate pBMN-CD4-Tailless. The
cytoplasmic domains of synaptotagmin I were amplified by PCR from the pBMN-Syt
1 plasmid to generate the following fragments: C2A-C2B (encoding residues
95-421), C2A (residues 95-265), C2B (residues 266-421), and CT (residues
393-421). Similarly, the cytoplasmic domains of synaptotagmin VII were
amplified by PCR from the pEGFP-Syt VIIs plasmid to generate the following
fragments: C2A-C2B (encoding residues 98-403), C2A (residues 98-260), C2B
(residues 261-403) and CT (residues 387-403). The forward primers were flanked
with a BstBI restriction site, and the reverse with a stop codon (for
the C2A fragments only) and a SalI restriction site. The PCR products
were digested and ligated to the corresponding sites of pBMN-CD4-Tailless to
generate in-frame CD4/synaptotagmin fusions. The Syt 7 C2A W253A, Syt 7 CT
W398A, and Syt 1 CT W404A mutants were generated using a QuikChange
site-directed mutagenesis kit (Stratagene). The C2B chimeras were generated in
a two-step PCR-based cloning strategy. First, the C2B fragment was amplified
using an outer primer complementary to a single synaptotagmin sequence and a
chimeric primer composed of half Syt 1 sequence and half Syt 7 sequence
surrounding the junction point. The corresponding C2B fragments were then
mixed together and amplified using the opposing synaptotagmin outer primers
alone to generate a chimeric C2B construct. The outer forward primers were
flanked with a BstBI restriction site, and the outer reverse with a
SalI restriction site. The PCR-generated chimeras were digested and
ligated to the corresponding sites of pBMN-CD4-Tailless. The chimeric
construct compositions are detailed in the
Fig. 7 legend. All constructs
were verified by sequencing.
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Transfections and retroviral infections
For retroviral infections, we used a vector derived from pBMN-Z-I-Neo in
which the LacZ gene was deleted (fragments
BamHI-SalI) and replaced by our genes of interest
(containing the CD4/synaptotagmin ORFs). The vector contains the internal
ribosome entry site of the encephalomyocarditis virus upstream of the neomycin
resistance gene. This permits both the gene of interest and the neomycin
resistance gene to be translated from a single bicistronic mRNA. Using this
method, nearly all surviving colonies will stably express the gene of interest
after selection with G418. Expression of the bicistronic mRNA is controlled by
the 5' viral LTR promoter (full-length Moloney LTR).
The Phoenix cells were transfected with the different CD4-synaptotagmin constructs using Fugene-6 transfection reagent (Roche). On transfection of the vectors, the Phoenix-packaging cell line produces replication-defective viral particles that were used for stable gene transfer and expression in PC12, CHO and NRK cells. Virus-containing supernatants were filtered through a low binding protein 0.45 µm filter (Pall Corporation) 48 hours after transfection, supplemented with 4 µg/ml of hexadimethrine bromide (Sigma-Aldrich) and used to infect PC12, CHO or NRK cells. After 48-72 hours, 400 µg/ml of G418 was added. Seven to 10 days after infection, colonies were pooled and propagated in culture in the presence of 400 µg/ml of G418. Cells were treated for 21 hours before the experiments with 500 nM of trichostatin A to enhance expression of the constructs.
Internalization assays using iodinated antibodies
50 µg of Q4120 antibody was iodinated on iodogen-coated tubes (Pierce
Chemical Co.) as described previously
(Clift-O'Grady et al., 1998).
Cells were plated on collagen and poly-D-lysine-coated 12-well plates two days
before the assay. Cells were incubated for 1 hour at 4°C with 100 ng/ml of
125I-Q4120 in DMEH-21 media supplemented with 1% BSA and 10 mM
HEPES, pH 7.4. Unbound antibody was removed by extensive washes. Cells were
next incubated at 37°C for 10 minutes to allow endocytosis and then
returned to 4°C. Antibodies remaining at the cell surface were removed by
two 10 minute acid-stripping washes at 4°C in PBS/BSA supplemented with 30
mM glycine and adjusted to pH 2.4. Acid-resistant antibody was collected by
lysing the cells in 2 M NaOH. The fraction of antibody internalized was
calculated by dividing the acid-resistant radioactive cpm by the sum of
acid-resistant and -accessible cpm and averaging over the samples taken in
triplicate. A background of acid-resistant counts in cells kept at 4°C was
subtracted from each value, and the error bars depict s.e.m.
Immunofluorescent microscopy
PC12 cells were plated onto eight-well collagen and poly-D-lysine-coated
slides at various densities two days before the slides were processed. For
uptake experiments, the cells were chilled on ice and were incubated for 1
hour at 4°C with 1 µg/ml anti-CD4 monoclonal antibody (Pharmigen, clone
RPA-T4) in DMEH-21 media supplemented with 1% BSA and 10 mM HEPES, pH 7.4.
Unbound antibody was removed by extensive washes. Cells were next incubated at
37°C for 10 minutes to allow endocytosis and then returned to 4°C.
After washing in ice-cold PBS, cells were fixed in 4% paraformaldehyde and
quenched in PBS, 25 mM glycine. Next, the cells were permeabilized and blocked
for 1 hour in 2% BSA, 1% fish skin gelatin and 0.02% saponin in PBS (blocking
solution). Finally, the cells were stained for 1 hour at room temperature with
secondary antibody, 10 µg/ml AlexaFluor 488 goat-anti-mouse IgG (Molecular
Probes) in blocking solution. After several washes, slides were mounted in
Movial and viewed with a 100x oil immersion lens on a Zeiss
Axioscope.
Flow cytometry analysis
PC12 cells stably expressing synaptotagmin VII C2A-C2B, C2A or CT were
transiently transfected with expression vectors using Lipofectamine 2000 (Life
Technologies). A single well of a six-well plate was transfected per sample
two days before flow cytometry analysis. The pIRES2-EGFP expression vectors
encoded either endocytic regulators [the wild-type or dominant negative
versions of dynamin (Schmidlin et al.,
2001) or eps15 (Benmerah et
al., 1999
)] or the regulatory fragment of interest of the C2B. In
the case of the eps15 constructs, the coding region was fused directly to
EGFP. However, in the case of the dynamin constructs and C2B fragments, the
vector also contains an internal ribosome entry site of the
encephalomyocarditis virus between the multiple cloning site (MCS) and the
enhanced green fluorescent protein (EGFP) coding region. Both vector
architectures permit the gene of interest (cloned into the MCS) and the
EGFP gene to be translated and allows for the identification, by flow
cytometry, of transiently transfected mammalian cells expressing EGFP and the
protein of interest. The cells were harvested, labeled at 4°C for 1 hour
with 1 µg/ml anti-CD4 monoclonal antibody (Pharmigen, clone RPA-T4) in PBS,
1% BSA. After washing, surface-bound antibody was visualized by addition at
4°C for 30 minutes of 1 µg/ml phycoerythrin-conjugated goat
F(ab')2 anti-mouse IgG antibody (Caltag) in PBS, 1% BSA.
After washing and resuspending in PBS, cells were analyzed with a Becton
Dickinson FACScalibur. The data were collected in a logarithmic mode, and the
mean of fluorescence intensity was calculated.
Structural analysis
The structural model of the synaptotagmin VII C2B was constructed using the
Swiss-Model program
(www.expasy.ch/swissmod/SWISSMODEL.html)
basing the homology-modeling on the published structure of synaptotagmin I C2B
(Fernandez et al., 2001). The
figure was then prepared using the WebLab Viewer Lite 3.2 software.
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Results |
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The C-terminal and C2A domains of synaptotagmin VII contain
functional internalization signals
Failure to be internalized is not an artifact of transfection, but is due
to the inaccessibility of an internalization domain. Synaptotagmin VII has
cytoplasmic domains very similar to those of synaptotagmin I, in particular
having the AP-2-binding site, the WHXL motif and the
calciumbinding/oligomerization residues
(Fig. 1C). To identify any
latent internalization signals, we generated fusion proteins that isolated the
individual cytoplasmic domains of synaptotagmin VII. Such a signal was
predicted to be in the C-terminal fragment of synaptotagmin VII (CT 7), a
segment of the C2B domain that includes the WHXL motif found to be a strong
internalization signal in synaptotagmin I (N. Jarousse, J. Wilson, D. Arac, J.
Rizo and R.B.K., unpublished). This region of synaptotagmin VII had as strong
an internalization signal as the corresponding domain in synaptotagmin I in
PC12 cells (Fig. 2A) and CHO
cells (data not shown). Likewise, the internalization of the synaptotagmin VII
CT was dependent on the presence of the analogous tryptophan within the WHXL,
as an alanine mutant failed to internalize at a rate above background in PC12
cells (Fig. 2B). Thus,
synaptotagmin VII has the same internalization motif as is used by
synaptotagmin I, but it is completely latent in PC12 cells.
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Next, the C2A domain of synaptotagmin VII (C2A 7) was tested for its
ability to be internalized in PC12 cells. Surprisingly, although the
corresponding domain had no endocytic properties in synaptotagmin I
(Blagoveshchenskaya et al.,
1999; Jarousse and Kelly,
2001a
), this domain in synaptotagmin VII contained a potent
internalization signal (Fig.
3A). Moreover, the signal within this domain appears to be a
universal signal, as demonstrated by the fact that C2A is internalized in CHO
cells as well (Fig. 3B). Notably, this domain, despite being homologous to the C2B domain, does not
contain the polybasic region known to bind AP-2. It does, however, contain a
sequence at its C-terminal end that is similar to the WHXL in location and
sequence. We next mutated the tryptophan in the C2A's WKXL sequence to an
alanine and tested this construct for its ability to be endocytosed. In this
case, the tryptophan is not involved in the endocytosis of this domain
(Fig. 3C).
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We verified the internalization of the C2A domain by a morphological assay of endocytosis. When PC12 cells were incubated with anti-CD4 antibody at 4°C, the labeled fusion proteins remained at the cell surface (Fig. 4A,C,E). When the temperature was increased to 37°C for 10 minutes, the CD4-C2A-C2B synaptotagmin VII construct continued to remain at the plasma membrane (Fig. 4B). However, when the synaptotagmin VII CT- and C2A-expressing cells were moved to 37°C, these proteins were taken up into internal compartments (Fig. 4D,F). This confirmed that both the CT and C2A domains of synaptotagmin VII contain strong internalization signals.
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The C-terminus and C2A domains are internalized by different
mechanisms
Endocytosis mechanisms can be subdivided according to their sensitivity to
dominant-negative inhibitors. Although the CT WHXL signal in synaptotagmin I
is not a typical internalization signal, it is internalized by a conventional
AP-2- and dynamin-dependent pathway (N. Jarousse, J. Wilson, D. Arac, J. Rizo
and R.B.K., unpublished). By analogy, the CT of synaptotagmin VII should
behave in a similar way. To substantiate this hypothesis, we used specific
inhibitors of traditional clathrin-mediated endocytosis to analyze their
effect on the trafficking of both the CT and C2A domains of synaptotagmin VII.
We used both functional and dominant-negative versions of dynamin or eps15,
tagged by coexpression with or fusion to GFP, transiently transfected into
stable cells expressing either the synaptotagmin VII CT fragment or the C2A
domain. The dynamin K44E mutant, mutated in its GTP-binding pocket, acted in a
dominant-negative fashion to inhibit dynamin-dependent endocytosis
(Herskovits et al., 1993).
Likewise, the eps15 fragments used were found to retain their endocytic
ability when the AP-2-binding domain was deleted (fragment D3
2) but
acted as dominant-negative inhibitors of AP-2-dependent endocytosis when the
EH domain was deleted (E
95/295)
(Benmerah et al., 1999
). We
measured both the cell surface CD4 staining for the synaptotagmin VII domains
and the GFP fluorescence for expression of the endocytic regulator by
fluorescence-activated cell sorting. In the event that the construct's
internalization was inhibited, we expected to see an increased amount of cell
surface staining in high-GFP-expressing cells. In fact we found that, in
agreement with the result for the synaptotagmin I CT, the CT of synaptotagmin
VII had increased levels at the plasma membrane when transfected with
dominant-negative dynamin in comparison with wild-type dynamin
(Fig. 5A). Furthermore, use of
the eps15 E
95/295 mutant also resulted in increased cell surface
expression of synaptotagmin VII CT compared to use of eps15 D3
2
(Fig. 5B). These conditions
potently inhibited internalization of the CT as the dominant negatives
resulted in a 6.8- and 2.0-fold increase in cell surface staining respectively
(Fig. 5E).
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When the parallel experiments were conducted using the synaptotagmin VII
C2A, the outcome was dramatically different. Specifically, neither the K44E
dynamin nor the eps15 E95/295 led to increased amounts of cell surface
synaptotagmin VII C2A (Fig.
5C-E). This places the internalization of synaptotagmin VII's C2A
domain into the unusual category of AP-2- and dynamin-independent
endocytosis.
A C2B subdomain is inhibitory to synaptotagmin VII endocytosis
Although the entire cytoplasmic domain cannot be internalized, the CT
region of synaptotagmin VII has the ability to be endocytosed when removed
from its normal environment as part of the C2B domain. When the structure of
synaptotagmin I's C2B domain was solved
(Fernandez et al., 2001), it
became apparent that the CT was an integral part of the eight-stranded
ß-sandwich structure of the C2B domain itself. We, therefore, asked if
the presence of the C2A domain affected recognition of the C2B's
internalization signal. In contrast to synaptotagmin I, the isolated C2B of
synaptotagmin VII did not retain an enhanced ability to be endocytosed
(Fig. 6). This suggests the
existence of a region within the C2B preventing internalization of
synaptotagmin VII by its tryptophan-based motif.
|
To more narrowly map the region within the C2B that was inhibitory to the
tryptophan-based motif, we designed chimeric constructs between the C2B
domains of synaptotagmin I and VII. Because these chimeras were being
generated within a single domain, we took care to choose chimeric junction
points that would least disrupt the domain's structure. Using the structure of
synaptotagmin I's C2B (Fernandez et al.,
2001) and the homology between the C2Bs of synaptotagmin I and
VII, we generated chimeric C2Bs where the transition points were within the
flexible loops, which were predicted to tolerate more motion than other
regions of the domain. Moreover, these switches were in areas of highly
conserved amino-acid sequence, so the resulting deformation should have been
minimal. Chimeras were made with ß-strands of the eight-stranded
ß-barrel replaced in pairs. The resulting chimeras fell into two
categories: a synaptotagmin VII C2B with increasing numbers of N-terminal
synaptotagmin-I-derived ß-strands, and the inverse series. These proteins
were subsequently tested for their ability to be endocytosed. The C2B series
with increasing contributions from synaptotagmin I clearly showed that when
the first two ß-strands of synaptotagmin VII were replaced with those of
synaptotagmin I, the inhibition was also removed
(Fig. 7A). Inversely, when the
C2B of synaptotagmin I gained the first two ß-strands of synaptotagmin
VII, it also gained the inhibitory property of synaptotagmin VII
(Fig. 7B). Therefore, this
inhibitory subdomain of synaptotagmin VII is transplantable and is in the
first two strands of the ß-sandwich. To test the possibility that the
synaptotagmin VII subdomain was binding to an inhibitory factor acting through
the C2B, we asked if the subdomain could be overexpressed to titrate out the
putative inhibitory factor. The prediction was that overexpression could
overwhelm the inhibitory machinery to activate endocytosis of synaptotagmin
VII. As a control, we also overexpressed GFP alone or the cognate region of
synaptotagmin I and then measured cell surface levels of synaptotagmin VII by
fluorescence-activated cell sorting. The overexpression of the inhibitory
fragment of synaptotagmin VII did not relieve the inhibition of endocytosis on
CD4-C2A-C2B synaptotagmin VII, as its levels at the cell surface remained high
(data not shown).
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Discussion |
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A paradox, however, is that synaptotagmin VII has internalization signals,
but they are latent. Synaptotagmin I has one of the internalization signals,
and it too is latent in cells that do not recycle synaptic vesicles. Synaptic
vesicle recycling is a form of compensatory endocytosis
(Jarousse and Kelly, 2001b) by
which the membranes of secretory vesicles are rapidly retrieved in a
homeostatic mechanism that preserves cell surface area. Because synaptotagmin
VII is involved in a number of regulated exocytosis events
(Caler et al., 2001
;
Gao et al., 2000
;
Gut et al., 2001
;
Martinez et al., 2000
;
Reddy et al., 2001
;
Shin et al., 2002
), one might
expect to find that its internalization signal is latent until exocytosis is
activated. We did not succeed in finding conditions or cell types in which
synaptotagmin VII is efficiently internalized. It is possible that
synaptotagmin VII's endocytic signals are only activated in situations where
the lysosomal pool is massively depleted. Similarly, a wound healing mechanism
might not automatically induce compensatory endocytosis until an activating
signal communicates completion of the healing process.
An additional interesting aspect of the internalization signals is the
unconventional nature of the C2A signal. We found that the internalization of
this domain did not require either dynamin or eps15, whereas the
internalization of synaptotagmin VII's CT fell into the more typical category
of dynamin- and eps15-dependent endocytosis. When IL-2 receptors are
internalized by an eps15-independent pathway
(Lamaze et al., 2001) they are
concentrated into detergent-resistant membrane subdomains that do not
colocalize with clathrin-coated pits. Thus, the C2A domain could allow
synaptotagmin VII to be internalized by an AP-2-independent pathway, which
might even allow targeting to a different intracellular compartment. However,
the route of internalization for IL-2 receptors is likely to be distinct from
that of the C2A of synaptotagmin VII since it appears to be dynamin dependent
(Lamaze et al., 2001
). In
contrast, some G-protein-coupled receptor (GPCR) family molecules have the
ability to be endocytosed in a dynamin-independent way
(von Zastrow, 2001
), but even
less is known about this pathway of endocytosis. It appears that synaptotagmin
VII C2A is internalized efficiently by a poorly described pathway that may not
involve clathrin. Furthermore, one intriguing possible explanation for the
latency of the internalization signals lies in these distinct modes of
internalization. It may be the case that the signals respond to different sets
of stimuli and have separate functions. For example, in the case of the m2
muscarinic acetylcholine receptor, the dynamin-dependent internalization is
agonist-induced, whereas the dynamin-independent internalization is continuous
(Pals-Rylaarsdam et al.,
1997
). Similarly, the two internalization signals in synaptotagmin
VII could be utilized in different contexts.
The latency of the tryptophan-based internalization signal within
synaptotagmin VII is unusual because it remains concealed owing to active
inhibition by a dominant subdomain of the C2B. Other cell surface proteins
also have latent endocytic signals. In the case of GPCRs, the receptors remain
at the cell surface until they are activated by ligand binding. This binding
triggers receptor phosphorylation and subsequent recruitment of
ß-arrestin to bridge the molecule to the endocytic machinery
(Ferguson, 2001). Likewise, for
receptor tyrosine kinases, ligand binding causes the receptor to become
covalently modified by autophosphorylation and, in some cases, ubiquitination
before being recognized by the endocytic machinery
(Clague and Urbe, 2001
). The
common feature of these endocytic events is that the signaling molecules must
first undergo a covalent modification that allows recruitment of machinery to
couple the signaling event to the trafficking event. At the synapse, endocytic
proteins are dephosphorylated after calcium-dependent transmitter release to
allow them to participate in endocytosis associated with synaptic vesicle
recycling (Lauritsen et al.,
2000
; Slepnev et al.,
1998
). In addition, synaptotagmin I itself undergoes
calcium-dependent phosphorylation by CaMKII, which strengthens its association
with the exocytic SNARE machinery of syntaxin and SNAP 25; subsequent
dephosphorylation weakens this interaction and could potentially promote
endocytosis of synaptotagmin I (Verona et
al., 2000
). This cyclic regulation of exocytic and endocytic
versions of synaptotagmin is supported by the fact that the phosphorylated
form of synaptotagmin I is found in exocytosis-competent synaptic vesicles but
not in clathrin-coated vesicles that presumably have arisen by endocytosis
(Hilfiker et al., 1999
). It is
possible that the C2B of synaptotagmin VII could exert its inhibitory effects
by either activating the phosphorylation of synaptotagmin VII or by preventing
its later dephosphorylation. Because of the relative proximity of the
inhibitory region to the WHXL motif (Fig.
7C), a phosphorylation event in the inhibitory region could cause
a conformational change that would render the WHXL inaccessible.
Alternatively, recruitment of a protein factor to this region could sterically
prevent recognition of the WHXL by the endocytic machinery. Inhibition by
phosphorylation would, however, require phosphorylation sites within the C2B
domain itself, whereas all previously identified sites are in the C2A
domain.
Synaptotagmin VII endocytosis is governed by a complex collection of activating and inhibitory signals that, under most conditions, retain synaptotagmin VII at the plasma membrane. It is our model that synaptotagmin VII acts at this site as a passive facilitator of endocytosis where it activates endocytosis of cargo molecules other than itself. Under certain, as yet unidentified, conditions, it can be internalized by two different endocytic pathways. Neuronal synaptotagmins have adapted regulated endocytosis for use in the efficient recycling of synaptic vesicle membranes. We hypothesize that a similar activating event takes place to make synaptotagmin VII's signals available for endocytosis in a context-dependent fashion.
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