Download Rabphilin mutants defective for Rab3 binding

3579
Journal of Cell Science 112, 3579-3587 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0526
High affinity Rab3 binding is dispensable for Rabphilin-dependent
potentiation of stimulated secretion
Gérard Joberty1,*, Paul F. Stabila2, Thierry Coppola3, Ian G. Macara1 and Romano Regazzi3
1Markey Center for Cell Signalling, Health Sciences Center, University of Virginia, Charlottesville, VA 22908,
2Department of Pathology, University of Vermont College of Medicine, Burlington, VT 05405-0068, USA
3Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Switzerland
USA
*Author for correspondence (e-mail: [email protected])
Accepted 5 August; published on WWW 30 September 1999
SUMMARY
Rabphilin is a protein that associates with the GTP-bound
form of Rab3, a small GTPase that controls a late step in
Ca2+-triggered exocytosis. Rabphilin is found only in
neuroendocrine cells where it co-localises with Rab3A on
the secretory vesicle membrane. The Rab3 binding domain
(residues 45 to 170), located in the N-terminal part of
Rabphilin, includes a cysteine-rich region with two zinc
finger motifs that are required for efficient interaction with
the small GTPase. To determine whether binding to Rab3A
is necessary for the subcellular localisation of Rabphilin,
we synthesised point mutants within the Rab3-binding
domain. We found that two unique mutations (V61A and
L83A) within an amphipathic α-helix of this region abolish
detectable binding to endogenous Rab3, but only partially
impair the targetting of the protein to secretory vesicles in
PC12 and pancreatic HIT-T15 cells. Furthermore, both
mutants transfected in the HIT-T15 beta cell line stimulate
Ca2+-regulated exocytosis to the same extent as wild-type
Rabphilin. Surprisingly, another Rabphilin mutant, R60A,
which possesses a wild-type affinity for Rab3, and targets
efficiently to membranes, does not potentiate regulated
secretion.
High affinity binding to Rab3 is therefore dispensable for
the targetting of Rabphilin to secretory vesicles and for the
potentiation of Ca2+-regulated secretion. The effects of
Rabphilin on secretion may be mediated through
interaction with another, unknown, factor that recognizes
the Rab3 binding domain.
INTRODUCTION
PC12 cells (Yamaguchi et al., 1993). Rabphilin is only found
in neuroendocrine cells (Shiritaki et al., 1993), and one splice
variant has been described (Chung et al., 1995). Rabphilin colocalises with the membrane-bound fraction of Rab3A on
dense-core secretory granules in both PC12 and chromaffin
cells (Chung et al., 1995; Shirataki et al., 1994).
Rab3A belongs to the Rab/YPT group of the Ras
superfamily (for reviews, see Valencia et al., 1991; Macara et
al., 1996). Rab proteins are involved in vesicular transport and
may regulate the vesicle docking step prior to membrane fusion
(Mayer and Wickner, 1997; Rybin et al., 1996). Rab proteins
are located in specific intracellular compartments (for review
see Simons and Zerial, 1993). Rab3A is expressed only in cells
that are capable of regulated secretion (Olofsson et al., 1988;
Burstein et al., 1989; Fischer von Mollard et al., 1990; Darchen
et al., 1995; Regazzi et al., 1996). Three additional isoforms of
Rab3 have been described (Matsui et al., 1988; Baldini et al.,
1992). In brain, Rab3C presents a localisation similar to that
of Rab3A with, however, a more heterogeneous distribution
among synapses (Fischer von Mollard et al., 1994; Li et al.,
1994; Castillo et al., 1997). Rab3B and Rab3D are expressed
in many different tissues (Baldini et al., 1992; Weber et al.,
1994; Regazzi et al., 1996).
Rabphilin is a 78 kDa protein identified by its ability to interact
with the GTP-bound form of the small GTPase Rab3A
(Shiritaki et al., 1993; McKiernan et al., 1993). The amino acid
sequence of the protein defines three regions (Kishida et al.,
1993). The N-terminal region contains the Rab3 binding
domain (amino acids 45 to 170) that includes a cysteine-rich
sequence with two zinc fingers (McKiernan et al., 1996; Stahl
et al., 1996; Ostermeier and Brunger, 1999). In a previous
study, we showed that the binding of two Zn2+ ions was
necessary but not sufficient for efficient binding of Rabphilin
to Rab3A (McKiernan et al., 1996). The central region contains
multiple consensus sequences for phosphorylation by protein
kinases, and Rabphilin is an in vitro substrate for protein kinase
A (Fykse et al., 1995; Lonart and Südhof, 1998). The Cterminal region contains two C2 domains, related to those of
protein kinase C and synaptotagmin, that can bind
phospholipids in a Ca2+-dependent manner (Chung et al.,
1998). Despite the fact that Rabphilin lacks a transmembrane
domain or consensus sequences for lipidic modifications, the
protein is mainly bound to membranes. The C2 domains are
required for efficient membrane attachment of Rabphilin in
Key words: Rab3 GTPase, Rabphilin3A, Ca2+-regulated secretion
3580 G. Joberty and others
The nucleotide-bound state of Rab3A is regulated by a
GTPase activating protein (Rab3A-GAP), exchange factors
(MSS4, Rab3-GRF, Rab3-GEP) and by binding proteins
such as Rab-GDI, Rim and Rabphilin (Burstein et al., 1991;
Fukui et al., 1997; Burton et al., 1993; Burstein and Macara,
1992; Wada et al., 1997; Matsui et al., 1990; Oishi et al.,
1998; Wang et al., 1998). Rabphilin itself inhibits the
GTPase activity of Rab3A promoted by Rab3-GAP and
possesses a weak nucleotide exchange activity toward
Rab3A. The physiological significance of these activities is
not known (Kishida et al., 1993). Another protein, Rabin3,
interacts with Rab3 proteins but its function is still unknown
(Brondyk et al., 1995).
When overexpressed in chromaffins cells, Rab3A inhibits
DMPP-induced, Ca2+-regulated secretion (Holz et al., 1994;
Chung et al., 1997) while Rabphilin potentiates secretion
(Holz et al., 1994; Chung et al., 1995). Fragments of
Rabphilin lacking one or both C2 domains, however, act as
potent dominant inhibitors of secretion (Chung et al., 1995).
Microinjection of the isolated N- or C-terminal parts of
Rabphilin also inhibit Ca2+-triggered cortical granule
exocytosis in metaphase II mouse eggs at fertilization
(Masumoto et al., 1996). These results, with others (Johannes
et al., 1994, 1996), suggest that Rab3A may form an
inhibitory complex with Rabphilin. Dissociation would
release Rabphilin and allow it to interact with other factors
leading to fusion and secretion of the vesicular cargo. Recent
studies suggest a particular involvement of Rab3A in
neuronal responses to repetitive stimulation. Long-term
potentiation is a synaptic plasticity phenomenon resulting in
a stable increase in synaptic transmission following repetitive
activation of excitatory synapses. This process is abolished at
hippocampal mossy fibre synapses in transgenic mice lacking
Rab3A (Castillo et al., 1997). These mice also show impaired
responses to repetitive stimuli (Geppert et al., 1994). In a
similar way, the microinjection of antisense oligonucleotides
directed against rab3A messengers in chromaffin cells
increases the response to repetitive stimulations, when a
desensitisation phenomenon is observed in the control
population (Johannes et al., 1994). In the Rab3A deficient
mice, the size of the readily releasable pool of vesicles is
normal, but Ca2+-triggered fusion is altered with a greater
number of exocytotic events occurring immediately following
the nerve impulse (Geppert et al., 1997). These results
suggest that Rab3A may regulate a late step in synaptic
fusion.
One function of Rab3A may be to recruit Rabphilin to the
secretory vesicle membrane, where it can interact with other
factors after its release from Rab3A. This hypothesis has
received experimental support (McKiernan et al., 1996; Stahl
et al., 1996), although our study suggested that factors other
than Rab3A may contribute to Rabphilin targetting
(McKiernan et al., 1996).
To determine the importance of the Rab3 binding domain of
Rabphilin for targetting, we synthesised point mutants within
the N-terminal region of that domain. We show that two point
mutants within an amphipathic α-helix abolish detectable
interaction with endogenous Rab3 but the proteins still partly
co-localise with Rab3A in PC12 cells and enhance Ca2+regulated secretion in pancreatic beta cells. Interestingly, a
third point mutant, which did not interfere with the binding of
Rab3A, nonetheless abrogated the ability of Rabphilin to
potentiate secretion.
MATERIALS AND METHODS
Synthesis of Rabphilin point mutant cDNAs
Point mutants in Rabphilin were created by the PCR-based
megaprimer method (White, 1993) using the bovine Rabphilin
sequence. PCR products were digested and subcloned into pGEX-2T
(Pharmacia) to express glutathione-S-transferase(GST)-fusion
proteins in bacteria; and into eukaryotic expression vectors pKH3 and
pK-GFP (McKiernan et al., 1996), to express proteins preceded by a
triple epitope hemaglutinin1 (HA1)-tag or fused with the green
fluorescent protein, respectively. Mutations were confirmed by DNA
sequencing. Cloning and expression of Rab3A as well as the
purification and cleavage of GST fusion proteins were performed as
previously described (McKiernan et al., 1996).
Rabphilin-Rab3A binding assay
Recombinant Rab3A cleaved from GST was loaded with [γ-32P]GTP
(10 µCi/ml at 5 mCi/nMol) in 20 mM Hepes, pH 7.4, 150 mM NaCl,
1 mM DTT, 2 mM EDTA, pH 8.0, 0.2 mg BSA/ml) and the nucleotide
was trapped on the protein by addition of MgCl2 to 10 mM. Counts
associated with Rab3A were determined by nitrocellulose filter
binding after extensive washing (McKiernan et al., 1996). Fifty
pmoles of GST-Rabphilin (1-206) fusion protein were bound to 5 µl
of glutathione-Sepharose beads in TBS (20 mM Tris-HCl, pH 7.4, 150
mM NaCl). The beads were washed 3 times in TBS and resuspended
in binding buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 0.2 mg/ml BSA and 1 mM GTP) such that the final reaction
volume was 100 µl. An equal amount of radiolabelled Rab3A-GTP
was then added to each tube and the mixture was incubated on ice for
10 minutes. In preliminary experiments, maximal binding was
observed within 5 minutes; negligible [32P]GTP release from Rab3A
was detected over a period of 15 minutes after initial mixing, either
in the absence or presence of Rabphilin. The bead suspension was
transferred to an Octavac 8-well strip attached to a vacuum apparatus.
Each well, fitted with a cellulose acetate filter (pore size 0.45 µm),
had been pre-rinsed with binding buffer to reduce non-specific
binding. Filters were washed and the relative amount of 32P-labeled
Rab3A bound to the beads in each well was determined by
scintillation counting.
Transfection of PC12 cells and immunofluorescence
PC12 cells were grown in DMEM (Gibco-BRL) supplemented with
5% fetal calf serum, 5% bovine serum, 5% horse serum, penicillin
(100 u/ml) and streptomycin (100 µg/ml). Transfection of PC12 cells
and immunofluorescence were carried out as previously described
(McKiernan et al., 1996). Briefly, cells were electroporated with a
total of 10-20 µg plasmid DNA per transfection at 280 mV/960 µF in
a Bio-Rad Gene Pulser. Cells were plated onto dishes or glass Labtek chamber slides (Nunc) coated with poly-L-lysine (Sigma).
Medium was changed after several hours and the cells were grown for
two days. Cells used for immunofluorescence were grown in the
presence of 40 ng recombinant human nerve growth factor (NGF, a
gift from Genentech) per ml to induce neurite outgrowths. For
immunofluorescence studies, cells were fixed in paraformaldehyde
(4%, w/v, in phosphate-buffered saline, PBS) and permeabilized with
cold methanol. GFP-Rabphilin fusion proteins were visualised
directly by the intrinsic fluorescence of the GFP(S65T). HA1-tagged
Rab3A was visualised using the 12CA5 anti-HA1 antibody and a Cy3linked goat anti-mouse antibody (Jackson Immunoresearch
Laboratories). Cells were imaged on a Bio-Rad MRC1000 confocal
microscope. To determine the relative degrees of overlap of the GFP
and Cy3 fluorescences within the transfected cells, an overlap quotient
Rabphilin mutants defective for Rab3 binding 3581
was calculated using the multiply function in the MPL software
package. This function multiplies the value for each pixel in one
image by the corresponding pixel in a second image. The result is then
reduced to a scale of 0-254. Low pixel values (<20) were deleted and
the remaining pixel values were summed and corrected for differences
in cell area, to give the overlap quotient for each pair of images
(McKiernan et al., 1996).
Partitioning of full-length Rabphilin mutants in cell
extracts
PC12 cell fractions were prepared as described (McKiernan et al., 1996).
HIT-T15 cell fractions were prepared in a similar manner, after lysing
the cells by sonication for 1 second. After SDS-PAGE and transfer on a
nitrocellulose membrane, proteins were submitted to immunoblotting
using a rabbit anti-GFP antibody (Clonetech) or the mouse monoclonal
12CA5 antibody and detected by chemiluminescence (KPL) using an
horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson
Immunoresearch Laboratories).
Binding of endogenous Rab3C from PC12 cell extracts to
GST-Rabphilin (1-206)
PC12 extracts were prepared as follows. All steps were performed
at 4°C. Cells were washed in PBS and lysed in lysis buffer (50 mM
Hepes, 150 mM NaCl, pH 7.4, 5 mM KCl, 0.5% v/v Triton X-100,
2 mM MgCl2, 2 mM DTT, 1 mM PMSF, 25 µg/ml aprotinin and 25
µg/ml leupeptin). Lysates were centrifuged at 500 g for 2 minutes
to eliminate nuclei and unlysed cells and then at 100,000 g for 1
hour. Extracts were incubated 30 minutes with GMPPNP (1 mM) in
the presence of EDTA (5 mM). Supernatants were then
supplemented with MgCl2 to a final concentration of 12 mM. The
cell content of a 150 mm diameter plate was the input for each
binding assay. GST-GFP or GST-Rabphilin (1-206) proteins (10 µg)
were bound to glutathione-Sepharose beads and then added to the
supernatants for an incubation time of 1 hour with rocking. Beads
were washed three times in buffer containing 50 mM Hepes, pH 7.4,
150 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100, 2 mM DTT, 1
mM PMSF, and twice in the same buffer without Triton. Bound
proteins were eluted with 15 to 20 mM glutathione (pH 8.0-8.5).
Eluted proteins were subjected to SDS-PAGE and then transferred
to nitrocellulose. Rab3C was detected using a specific, anti-Rab3C
antibody. This rabbit anti-peptide antibody, made against unique
residues of Rab3C (amino acids 195 to 209), does not recognise
Rab3B or Rab3D, and shows only very weak cross-reactivity with
Rab3A (G. Joberty and A. Zahraoui, unpublished data). GST fusion
proteins were detected with a monoclonal antibody against GST
(Santa Cruz). HRP-linked secondary antibodies (Jackson
ImmunoResearch Laboratories) were used to visualise proteins by
chemiluminescence.
Secretion from transfected HIT-T15 cells
HIT-T15 cells were cultured as described (Regazzi et al., 1990). On
the day of the experiment, 3×106 cells, were resuspended in 300 µl of
serum-free RPMI 1640 and were electroporated in the presence of 30
µg of a plasmid encoding human growth hormone (hGH) and 30 µg
of the plasmids encoding the full-length Rabphilin constructs.
Immediately after transfection the cells were diluted in culture
medium and seeded in 24 multiwell plates. Three days after
transfection the cells were pre-incubated during 30 minutes in 20 mM
Hepes, pH 7.4, 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 10 mM
glucose and 1 mM MgCl2. The medium was then aspirated and the
cells stimulated for 10 minutes with the same buffer but containing
53 mM NaCl and 80 mM KCl. Exocytosis from transfected cells was
determined by measuring by ELISA (Boeringer Mannheim) the
amount of hGH secreted into the medium during the incubation
period. To verify the expression of the HA-Rabphilin proteins the cells
used in the secretion experiments were collected in SDS buffer and
analyzed by western blotting using the anti-HA antibody. Targetting
of the HA-Rabphilin proteins in HIT-T15 cells was assessed by
confocal microscopy using the anti-HA antibody to label the
transfected proteins and a polyclonal anti-insulin antibody to label the
secretory vesicles (Iezzi et al., 1999).
RESULTS
Residues within the N-terminal domain of Rabphilin
are critical for association with Rab3 proteins
Analysis of the N-terminal, conserved Rab3 binding domain of
Rabphilin using the coiled-coil algorithm of Lupas et al. (1991)
showed that this region contains a putative α-helix (amino
acids 44 to 90, Fig. 1, top). A projection along the axis of this
helix reveals a distinct amphipathic bias (Fig. 1, bottom). The
structural prediction has been recently confirmed by the
publication of the crystal structure of a Rab3A-Rabphilin
complex (Ostermeier and Brunger, 1999). To identify residues
important for Rab3 binding we mutated to alanines a number
of conserved amino acid residues within the putative α-helix.
Polar (basic residues R60 and R78, acidic residues D81 and
D84) as well as aliphatic amino acids (L83 and V61) were
chosen.
Mutated GST fusion proteins of Rabphilin (1-206) were
attached to GSH-Sepharose and assayed for binding to
recombinant Rab3A:[γ-32P]GTP. The Rabphilin (1-206)
fragment was used because it is resistant to proteolysis and has
been shown to bind Rab3A with the same affinity as the fulllength protein (McKiernan et al., 1996). Mutations of the polar
amino acids did not noticeably affect the association with
Rab3A, but mutations of both aliphatic residues, L83A and
V61A, showed, respectively, a 30% reduction and an almost
complete abolition of the binding to Rab3A (Fig. 2).
To determine whether endogenous, prenylated Rab3
proteins would show the same differential interaction with the
mutants of Rabphilin, we incubated the GST-Rabphilin(1-206)
constructs attached on glutathione-Sepharose beads with
extracts of PC12 cells. GST-GFP (a fusion protein between
GST and the Green Fluorescent Protein) was used as a
negative control. Because Rabphilin interacts preferentially
with the GTP-bound form of Rab3A, the PC12 extracts were
incubated with GMPPNP, a slowly hydrolysable analogue of
GTP, prior to addition of the GST fusion proteins, to allow
conversion of endogenous Rab3 proteins to the active form.
The proteins retained by the beads were immunoblotted with
a specific anti-Rab3C antibody. We have confirmed that all
four Rab3 isoforms (A-D) bind with similar affinities to
Rabphilin (Chung et al., 1999). We checked that equal
amounts of fusion protein were bound to the beads using an
anti-GST antibody. As shown in Fig. 3, the wild-type
Rabphilin (1-206) and mutant R60A fusion proteins formed
stable complexes with Rab3C produced by PC12 cells, while
the mutant V61A and GST-GFP control did not. These data
confirm the results obtained with recombinant Rab3A.
However, Rab3C did not detectably bind to the L83A mutant
of Rabphilin(1-206). This difference is likely to be related to
the much lower concentration of Rab3C present in PC12
extracts, as compared to the assay in which recombinant
Rab3A was used.
The results demonstrate that certain hydrophobic residues
within a putative amphipathic α-helical region of the Rab3
3582 G. Joberty and others
Rab3 binding
domain
Fig. 1. Putative amphipathic α-helix
of Rabphilin Rab3-binding domain.
Top: schematic structure of
Rabphilin 3A protein showing the Nterminal Rab3 binding domain with
the two Zn2+ binding motifs. An
amphipathic α-helical region,
identified using the coiled-coil
algorithm of Lupas et al. (1991), is
located just upstream of these
motifs. Bovine and C. elegans
sequences of that region are aligned.
Identical amino acids among both
sequences are indicated via straight
lines and conserved ones by doubledots. Numbering corresponds to the
bovine sequence of Rabphilin.
Residues with numbers from
position 60 to 84 indicate amino acid
residues that were mutated to
alanines. Bottom: helical wheel
projection of residues 45-90
generated by the Protean protein
analysis program in the Lasergene
Software package (DNA-Star). The
amphipathic bias is revealed by the
presence of most aliphathic residues
(in black) on the right part on the
helix, whereas the charged amino
acids (in grey) are located primarily
on the other side. Arrows and
arrowheads show respectively
aliphatic and charged residues that
were mutated to alanines. Asterisks
show residues that interact directly
with Rab3A, as shown by the crystal
structure of the Rabphilin:Rab3A
complex (Ostermeier and Brunger,
1999).
N
C2
C
1 44
90
Zn Zn
170
C2
710
396
44
60 61
78
81
83 84
90
B. t.
QRKQEELTDEEKE I IN RV I ARAE KMEEMEQERIG R L V D RLENMRKNV
C. e.
K A Q T G S I T A A E Q E H I Q K V L A K A E E S K S K E Q Q R IG K M V D R L E K M R R R A
:
:
: :
:
:
:
:
: :
120
166
N
R
89
82
R
71
78
N
K
53
K
61
E
56
63
E
88
binding domain of Rabphilin are essential for high affinity
association with the small GTPase.
Rabphilin targetting is reduced but not abolished by
loss of Rab3 binding
To determine the role of Rab3 binding in targetting Rabphilin
to vesicle membranes, we compared by confocal microscopy
the cellular localisation with Rab3A of the R60A, V61A and
L83A mutants of full-length Rabphilin fused with GFP.
Vectors encoding the GFP-Rabphilin constructs were cotransfected into PC12 cells together with a vector encoding
the triple HA1-tagged Rab3A protein. As described
previously, HA3-Rab3 localises to vesicles concentrated at the
cell periphery and in the neurite extensions induced by
treatment of PC12 cells with NGF. Wild-type GFP-Rabphilin,
co-localises with the HA3-Rab3 (Fig. 4A). GFP-Rabphilin
(R60A), which binds Rab3 with wild-type affinity, was
detected mainly in the neurite outgrowths. In contrast, the
labelling of the V61A mutant, which can not bind Rab3A, was
more diffused. Nonetheless the protein still displayed
significant co-localisation with Rab3A, especially in the
neurite extensions. The L83A mutant, which has a reduced
*
*
E
A
D
51
48
N
K
59
55
E
G
I
T
E
I
*
62
58
*
*
65
I
E
*
V
90
72
54
47
52
70
L
79
Q
A
D
86
V
L
50
49
E
74
81
K
E
E
*
68
57
46
67
M
M
I
E
60
*
75
64
R
85
R
R*
M
L
83
76
69
66
Q
77
E
73
V
80
R
87
84
affinity for Rab3 in vitro, gave an intermediate phenotype.
Immunofluorescence images were quantitated as previously
described (Mc Kiernan et al., 1996; see also Materials and
Methods). As shown in Table 1, the overlap quotient for the
mutant R60A (0.136) is essentially the same as for wild-type
GFP-Rabphilin (0.127), indicating a high degree of colocalisation with HA3-Rab3. The partial co-localisation of the
mutant V61A with HA3-Rab3A is reflected by an overlap
quotient (0.077) that is intermediate between those of wildtype GFP-Rabphilin and of free GFP (0.018). The overlap
quotient between mutant L83A and Rab3A is close to that of
mutant V61A (0.095). Localisation studies were also
performed using a pancreatic beta cell line, HIT-T15. These
cells accumulate insulin within secretory granules and have
been used previously to study the effects of Rabphilin on
stimulated exocytosis (Arribas et al., 1997). Confocal
microscopy demonstrated a high degree of co-localisation
between insulin-containing granules and HA-Rabphilin (fulllength) in cells transfected with wild-type or with the R60A
or V61A mutant Rabphilin proteins (Fig. 4B).
Fractionation studies were performed on extracts of both
PC12 and HIT-T15 cells co-expressing GFP-Rabphilins
Binding of point mutants of
GST-Rabphilin(1-206) to Rab3A
(% of wt Rabphilin(1-206) binding)
Rabphilin mutants defective for Rab3 binding 3583
Table 1. Quantitation of co-localisation of HA1-Rab3A
and GFP-Rabphilin proteins in transiently co-transfected
PC12 cells
125
100
75
*
50
25
**
0
wt
R60A V61A R78A D81A L83A E84A
Rabphilin(1-206) proteins
Fig. 2. Identification of residues within the Rab3 binding domain of
Rabphilin that are important for Rab3A binding. GST-Rabphilin
fusion proteins were attached to glutathione-Sepharose beads and
incubated with [γ-32P]GTP-Rab3A. The beads were washed and the
amount of associated Rab3A was determined by scintillation
counting. Binding is expressed as percentage relative to GSTRabphilin(1-206). Binding data are the means of two experiments
performed in duplicate. Non-specific binding obtained with the GST
protein alone (less than 2%) was deducted from all other values. The
range for each experiment was ≤ ±1%.
(full-length) with HA3-Rab3. As expected, based on
previous data (McKiernan et al., 1996), unmutated GFPRabphilin was detected predominantly in the membrane
fraction of PC12 cell lysates (Fig. 5A). The co-expression of
HA3-Rab3A slightly increased the proportion of membranebound GFP-Rabphilin. The distribution of the R60A
Rabphilin mutant was similar to that of the wild-type
protein. Consistent with the confocal imaging, the two point
mutants with reduced or negligible binding to Rab3 (V61A,
L83A) exhibited a small increase in the proportion of GFPRabphilins in the soluble fraction (Fig. 5A). Similar
experiment was performed using HIT-T15 cells expressing
full-length HA3-Rabphilins (wild-type or point mutants
forms). Rabphilin was more abundant in the cytosol of these
Fig. 3. Mutations to alanine of aliphatic residues 61 and 83 of
Rabphilin abolish the binding to endogenous Rab3 proteins. GSTGFP or GST-Rabphilin(1-206) were bound to glutathione-Sepharose
beads and incubated with PC12 extracts in which the GTP-binding
proteins were previously loaded with GMPPNP. After washing, GST
fusion proteins and Rab3C were analysed by immunoblotting using,
respectively, a monoclonal anti-GST antibody and a polyclonal antiRab3C antibody. Lane 1, corresponds to an aliquot (10% of input) of
the PC12 extract and is used as a negative control for the anti-GST
antibody and as a positive control for the Rab3C antiserum.
Red channel
Green channel
HA3-Rab3A
HA3-Rab3A
HA3-Rab3A
HA3-Rab3A
HA3-Rab3A
GFP-Rabphilin (wild-type)
GFP
GFP-Rabphilin R60A
GFP-Rabphilin V61A
GFP-Rabphilin L83A
Overlap quotient
± s.e. (n≥5)
0.127±0.009
0.018±0.005*
0.136±0.016
0.077±0.010**
0.095±0.014
*P value ≤0.01 (difference very significant)
**P value ≤0.001 (difference extremely significant)
Overlap immunofluorescence quotients were determined by dividing the
number of pixels in the multiplied image by the sum of pixels in the two
original images as described in Material and Methods. A minimum of five
cells were analysed per transfection condition. P values were calculated by an
unpaired t-test as compared to the overlap quotient for pKH3-Rab3A and
GFP-Rabphilin (wild-type) to determine if there was a significant difference
in the overlap quotient.
cells in comparison with the PC12 cells (Fig. 5B). Replacing
the GFP tag by a HA3 tag did not change significantly the
results of the partitioning in PC12 cells (data not shown), and
the difference may therefore be cell-specific. Similar to the
PC12 cells, the distribution of the V60A mutant in HIT-T15
cells was close to those obtained with the wild-type protein,
whereas both mutants V61A and L83A, that do not bind
Rab3 with a high affinity, showed a slightly higher
accumulation in the soluble fraction (Fig. 5B).
We conclude that the effects of the mutations or targetting
are independent of cell type. The results indicate that high
affinity binding to Rab3A is required for efficient targetting
of Rabphilin to secretory vesicle membranes, but that
targetting is still possible in the absence of detectable Rab3A
binding.
Rabphilin mutants that do not bind Rab3 are capable
of enhancing evoked secretion
To assess how the different mutants of Rabphilin affect Ca2+regulated secretion we chose to test the effect of the Rabphilin
mutants on stimulated exocytosis of HIT-T15. HIT-T15 cells
were transiently co-transfected with each of the constructs and
with the gene encoding hGH. Ectopically expressed hGH is
targeted to dense core insulin-containing secretory granules (T.
Coppola and R. Regazzi, unpublished observation). Consistent
with previous studies, we observed that ectopic expression of
full-length Rabphilin enhances K+-stimulated hGH secretion
(Fig. 6A). Surprisingly, however, the two mutants of Rabphilin
that do not bind efficiently to Rab3, V61A and R83A,
stimulated secretion to comparable extents (54% and 65%,
respectively). In contrast, the R60A mutant, which binds
Rab3A with an efficiency undistinguishable from that of wildtype Rabphilin and is targeted to the secretory vesicle
membrane, did not enhance regulated secretion. This lack of
effect was not a consequence of reduced expression of the
R60A mutant, as determined by immunoblotting (Fig. 6B). To
determine whether the loss of function was specific for the
mutation in position 60, we tested two others mutants, R78A
and D81A, located on the same side of the amphipathic helix.
As was shown in Fig. 2, both mutants are able to interact
normally with Rab3A; in HIT-T15 cells, they both show a
partitioning similar to those obtained for the wild-type and
3584 G. Joberty and others
Fig. 4. Localisation of GFP-Rabphilin point
mutants in PC12 and HIT-T15 cells by confocal
microscopy. (A) PC12 cells were transiently cotransfected with plasmids encoding HA3-Rab3A
and either GFP, GFP-Rabphilin full-length wildtype or GFP-Rabphilin full-length point mutants
(R60A, V61A or L83A). Cells were treated and
processed for immunofluorescence as described
in Material and Methods. All GFP-Rabphilin
constructs show significant co-localisation with
HA3-Rab3A, as shown with the overlap between
the two channels (A, right panel). (B) Cells of
the pancreatic beta cell line HIT-T15 were
transfected with plasmids encoding either HA3tagged wild-type (wt) Rabphilin or HA3-tagged
Rabphilin mutants (R60A) or (V61). After three
days, the cells were double-labelled with an
antibody against insulin and with the antibody
against the HA3 tag and were analysed by
confocal microscopy.
V60A mutant proteins (Fig. 5B). We found that the R78A and
D81A mutants potentiated K+-stimulated hGH secretion as
well as wild-type Rabphilin (Fig. 6A).
DISCUSSION
We have identified two point mutations in the N-terminal
region of Rabphilin that abolish (V61A) or substantially reduce
(L83A) binding to recombinant Rab3A and to endogenous
Rab3 from PC12 cells extracts. Both mutations affect
hydrophobic residues that are conserved between the
mammalian Rabphilin and its putative homolog in the
nematode C. elegans (Wilson et al., 1994). Four other
mutations of charged amino-acid residues on the opposite face
of the predicted α-helix and that are not conserved throughout
evolution, had no effect on Rab3 binding. The recently
published crystal structure of the Rabphilin-Rab3A complex
confirms that this region forms an extended α-helix that
interacts with Rab3A (Osteimeier and Brunger, 1999). The
structure clearly shows that amino acid residue V61 contacts
directly the C-terminal region of Rab3A whereas the adjacent
residue, R60, does not, accounting for the differences in
binding that we observed. The reason why the mutation of
residue L83 strongly reduces Rab3 binding is less obvious. The
structural data suggest that a mutation in that position might
modify either the interaction between α-helices 1 and 2 or the
Rabphilin mutants defective for Rab3 binding 3585
Fig. 5. Partitioning of Rabphilin constructs in PC12 and
HIT-T15 cells. (A) GFP-Rabphilin full-length proteins
were transiently co-expressed with HA3-Rab3A in PC12
cells. GFP-Rabphilin (wild type) was also co-transfected
with the pKH3 empty vector (two first lanes). (B) HA3Rabphilin full-length proteins were transiently expressed
in HIT-T15 cells. (A and B) Cell lysates were partitioned
into soluble and particulate fractions and were analysed
by immunoblotting using a monoclonal anti-GFP (A) or
monoclonal anti-HA (B) antibodies, as described in
Material and Methods. Immunoblots were scanned with
a densitometer to determine relative amounts in each
fraction.
structure of the downstream zinc binding domain. It is unlikely
that the V61A or L83A mutations cause large structural
perturbations. Significant fractions of the mutant proteins are
targeted to the correct membrane in two different cell types and
the mutations in Rabphilin did not cause reduced expression,
detectable proteolytic degradation or targetting to lysosomes.
These effects would be a likely consequence of protein
misfolding. Finally, both mutants remained able to potentiate
Ca2+ regulated secretion.
It has been proposed that Rabphilin is recruited to synaptic
vesicles by Rab3A/C (Li et al., 1994), via a mechanism similar
to the recruitment by Ras of the Raf1 kinase (Stahl et al., 1996).
Our data suggest that the mechanism permitting the
recruitment of Rabphilin is more complicated, since binding to
Rab3 is not an absolute requirement for targetting secretory
vesicles of PC12 or HIT-T15 cells. In some cell types,
Fig. 6. K+-stimulated hGH release from HIT-T15 cells expressing the
HA3-tagged Rabphilin constructs. (A) HIT-T15 cells were cotransfected with a plasmid encoding for hGH and either with empty
pKH3 vector or plasmid encoding for HA3-tagged wild-type (wt)
Rabphilin or for one of the different HA3-Rabphilin point mutant
(R60A, V61A, R78A, D81A or L83A). In each case basal
unstimulated secretion of hGH was measured and deducted from K+stimulated secretion. Stimulated secretion obtained for cells
transfected with the empty pKH3 vector was defined as 100% of K+stimulated exocytosis. * difference with control (Cont.) is
statistically significant (P<0.01, Student’s t-test). (B) As a control for
expression levels, HIT-T15 cells were transfected with empty vector
or with the indicated HA3-tagged Rabphilin constructs. Three days
later the cells were lysed and analysed by western blotting using the
12CA5 antibody directed against the HA3 tag.
Rabphilin seems to be unstable when not bound to Rab3A.
Neurons from genetically-engineered Rab3A−/− mice, for
example, express substantially reduced levels of Rabphilin.
Similarly, the Rabphilin mutants that are defective in Rab3
binding (V61A, L83A) could not be expressed in primary
chromaffin cells (R. Holz, unpublished observation). One
B
HA-
3586 G. Joberty and others
interpretation of these data is that undocked Rabphilin is
rapidly proteolysed. This problem of unstability did not occur
in either PC12 or HIT-T15 cells, perhaps because, in these cell
types, proteins other than Rab3 can promote docking to the
vesicle membrane. Accordingly, Rabphilin can bind to synaptic
vesicles in which Rab3A has been biochemically stripped
(Shirataki et al., 1994).
Apart from mutant R60A, all Rabphilin mutants stimulated
exocytosis regardless of their ability to bind Rab3, arguing that
the interaction is not required for this function. It can not be
completely ruled out that mutants V61A and R83A are able to
bind Rab3 in vivo but this interpretation is unlikely. First, these
mutants were unable to pull down endogenous Rab3C from
PC12 extracts, although wild-type Rabphilin shows similar
affinities for all Rab3 proteins. Second, residue 61 is now
known to directly interact with Rab3A (Ostermeier and
Brunger, 1999). A previous study in a comparable system also
suggested that the Rab3 binding domain was not required for
Rabphilin to potentiate regulated secretion (Arribas et al.,
1997). This study was made using N-terminal deletions of
Rabphilin. Here, we show that unique point mutations within
full-length Rabphilin can considerably impair Rab3 binding,
and that a significant fraction of these proteins is correctly
targeted and still potentiates Ca2+-dependent exocytosis in
pancreatic beta-cells. Remarkably, another point mutant,
R60A, was unable to potentiate exocytosis. This mutant was
expressed and targeted to secretory vesicles to a degree similar
to that of the V61A and L83A mutants. This result suggests
that multiple factors bind to the N-terminal region of Rabphilin
and regulate its function. The conclusion that Rabphilinmediated effects on exocytosis are independent of Rab3A
binding (Arribas et al., 1997; this study) are supported by an
independent recent report in which a series of mutants of
Rab3A were used (Chung et al., 1999). The authors found that
there is no correlation between the binding of Rab3A to
Rabphilin and the ability of the Rab3A effector-domain
mutants to inhibit Ca2+-dependent exocytosis in primary
chromaffin cells. In particular, Rab3A mutants T54A and F59S
do not efficiently bind Rabphilin but do inhibit K+-evoked
secretion like the wild-type Rab3A. Thus, the inhibitory effects
of ectopically-expressed Rab3A may be a consequence of
binding to and titrating out key factors other than Rabphilin
that are involved in the secretory response.
Overall, these data suggest that targetting of Rabphilin to the
secretory membrane and regulation of its function is more
complex than previously thought. Both the targetting of
Rabphilin to the secretory vesicle and its capability to
potentiate regulated secretion in different cell types are not
dramatically affected in the absence of high affinity Rab3A
binding. We propose that both events may occur in two
different ways: one dependent on Rab3 binding and another
that is independent of Rab3. Although Rab3 binding is
probably the predominant mechanism that localises Rabphilin
to the secretory vesicle membrane in neurons, interaction of
the protein with other proteins, via its proline rich regions or
C2 domains, may attain the same goal. To potentiate secretion
evoked by K+-depolarisation, Rabphilin may need to bind other
protein(s); an interaction that would be impaired in the case of
the R60A mutant. There are several possible candidate
proteins. Rabphilin, via its binding to Rabaptin, an effector of
Rab5 (Stenmark et al., 1995), is known to inhibit receptor-
mediated endocytosis of transferrin in PC12 cells (Ohya et al.,
1998). Another protein, α-actinin, has been reported to interact
with the N-terminal region of Rabphilin (Kato et al., 1996).
The interaction stimulates the ability of α-actinin to cross-link
actin filaments in bundles, and this effect is inhibited by
GTPγS-Rab3A. However, the relationship of α-actinin binding
to vesicle docking and fusion is unknown. In any case, it
appears that the effects of Rabphilin and Rab3 on secretion are
separable and distinct, and that Rabphilin may not be the
effector through which ectopically-expressed Rab3 exerts its
inhibitory effect on exocytosis.
The authors thank Dr Paula Tracy, University of Vermont, for the
gift of purified thrombin and Véronique Perret-Menoud for technical
assistance. This work was supported by NIH grant CA56300 from the
National Institute of Health (DHHS) to I.G.M and by grant 3100050640.97 from the Swiss National Science Foundation to R.R. P.F.S.
was supported by an NIH Cancer Biology training grant
(T32CA09286).
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