Download M6PRs are found in a subset of PC12 cell ISGs

3955
Journal of Cell Science 112, 3955-3966 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0535
Differential distribution of mannose-6-phosphate receptors and furin in
immature secretory granules
Andrea S. Dittié1,*, Judith Klumperman2 and Sharon A. Tooze1,‡
1Secretory Pathways Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK
2University of Utrecht Medical School, Institute for Biomembranes, Heidelberglaan 100, 3584CX Utrecht, The Netherlands
*Present address: Bayer AG, Apratherweg 18a, D-42096 Wuppertal, Germany
‡Author for correspondence (e-mail: [email protected])
Accepted 1 September; published on WWW 3 November 1999
SUMMARY
In neuroendocrine cells sorting of proteins from immature
secretory granules (ISGs) occurs during maturation and is
achieved by clathrin-coated vesicles containing the adaptor
protein (AP)-1. We have investigated the role of
the mannose-6-phosphate receptors (M6PRs) in the
recruitment of AP-1 to ISGs. M6PRs were detected in ISGs
isolated from PC12 cells by subcellular fractionation, and
by immuno-EM labelling on cryosections. In light of our
previous results, where greater than 80% of the ISGs were
found to contain furin, we examined the relationship
between furin and M6PR on ISGs. By immunoisolation
techniques we find that 50% at most of the ISGs contain
the cation-independent (CI)-M6PR. Using sequential
immunoisolation we could demonstrate that there are two
populations of ISGs: those that have both M6PR and furin,
and those which contain only furin. Furthermore, using
immobilized GST-fusion proteins containing the
cytoplasmic domain of the CI-M6PR we have shown
binding of AP-1 requires casein kinase II phosphorylation
of the CI-M6PR fusion protein, and in particular
phosphorylation of Ser2474. Addition of these
phosphorylated GST-CI-M6PR fusion proteins to a cellfree assay reconstituting AP-1 binding to ISGs inhibits AP1 recruitment to ISGs.
INTRODUCTION
and consistent with the former model, in other cell types the
regulated secretory proteins represent a smaller percentage of
newly synthesised proteins and may have to be actively
selected by sorting receptors to be concentrated in nascent
secretory granules. Even less is known about the mechanisms
involved in the biogenesis of the future secretory granule
membrane surrounding the core proteins. The mature secretory
granule (MSG) membrane contains a wide variety of proteins,
for example the vacuolar H+-ATPase, cytochrome b561 and
synaptotagmin (for a recent comprehensive list see Apps,
1997), whose function, in many cases, is known but whose
trafficking into the MSG has not been extensively studied.
We have been characterizing the ISG in the neuroendocrine
cell line PC12 in an attempt to address some of the current
questions about the mechanisms of sorting of both the
secretory granule content and membrane during secretory
granule formation. ISGs are the intermediate vesicular
compartment between the TGN and MSGs (Tooze et al., 1991),
and were first identified in pituitary mammotroph cells by EM
autoradiography as being the initial site of hormone
concentration after the Golgi complex (Farquhar and Palade,
1981). Subsequent studies have added to the morphological
definition of this compartment in a variety of cell types (for
review see Tooze, 1991; Arvan and Castle, 1998). The
consensus from morphological and more recent biochemical
The molecular details of secretory granule formation from
trans-Golgi network (TGN) membranes is the subject of
controversy and the precise mechanisms involved in secretory
granule formation are not yet known. Regarding the selection
of secretory granule content proteins experimental data has
been obtained, using endocrine and neuroendocrine cell
systems, which supports two disparate, but not mutually
exclusive, models: (i) sorting is required for inclusion into
ISGs and occurs via an active process utilizing membrane
associated components, or (ii) sorting is not required for
inclusion into ISGs and does not occur prior to or during
budding from the TGN (for recent reviews see Tooze, 1998 and
Arvan and Castle, 1998). Reconciliation of the experimental
data is possible if one considers cell-type specific variations in
the amount of newly synthesized regulated secretory proteins.
The data to support the latter model is from cell types where
greater than 90% of all newly synthesised proteins are
delivered to the secretory granule. This large efflux into the
regulated pathway, which could be analogous to ‘bulk flow’
(Pfeffer and Rothman, 1987), may alleviate the need to
selectively sort in the TGN the regulated secretory proteins
from other soluble proteins; the latter may comprise only 10%
of the protein in the regulated secretory pathway. In contrast,
Key words: Regulated secretion, AP-1, Clathrin-coated vesicle,
Furin, Casein kinase II
3956 A. S. Dittié, J. Klumperman and S. A. Tooze
studies is that the ISG contains prohormones, has a mildly
acidic pH, and bears patches of a clathrin coat which is
comprised of adaptor protein (AP)-1 and clathrin unlike MSGs
which contain hormones, are acidic, and have no clathrin
patches. The protein composition of the ISG membrane is not
well characterized, however the cation-independent (CI)mannose-6-phosphate receptor (M6PR), the cation-dependent
(CD)-M6PR (Klumperman et al., 1998), and furin (Dittié et al.,
1997) occur in ISGs but not in MSGs. The properties of, and
differences between, ISGs and MSGs have contibuted to a
model in which maturation from an ISG to an MSG requires
removal of both soluble and membrane proteins. Secretion of
soluble C-peptide (derived from proinsulin through
endopeptidase cleavage) from ISGs occurs via a pathway
referred to as the ‘constitutive-like pathway’ (Kuliawat and
Arvan, 1992), while vesicles budding from the ISG have been
identified which are clathrin-coated and contain M6PRs
(Klumperman et al., 1998). It is not yet known whether these
pathways overlap, ie if the clathrin-coated vesicles also contain
C-peptide. Finally, it has not yet been determined if the M6PR
positive, clathrin coated membranes and vesicles also contain
furin.
The M6PRs and furin are found in TGN membranes,
endosomes and the plasma membrane, and appear to cycle
through these compartments (for review see Le Borgne and
Hoflack, 1998 and Molloy et al., 1999). The cytoplasmic
domains of M6PR and furin interact with either AP-1 in the
TGN, or AP-2 at the plasma membrane, to promote inclusion
of these proteins in clathrin-coated vesicles (CCVs) budding
from these compartments. Sequences such as the tyrosine
motif, the di-leucine motif and the acidic cluster (for review
see Kirchhausen et al., 1997) in the cytoplasmic domain of
the M6PRs are required for the TGN localization of the
M6PRs. Two of these sequence motifs (the tyrosine motif and
the acidic cluster) have also been shown to be required for
TGN localization of furin. Furthermore, experiments
performed with fibroblasts lacking M6PRs have confirmed
that the acidic cluster of amino acids, when phosphorylated
by casein kinase II (CKII), is important for the recruitment
of AP-1 to TGN membranes and the formation of CCVs
(Mauxion et al., 1996).
Our previous experiments have demonstrated that the
formation of a clathrin-coat on ISGs can be attributed to the
recruitment of AP-1 by the cytoplasmic domain of furin.
Importantly, furin present on the ISGs must be phosphorylated
by casein kinase II (CKII) in order to bind AP-1 (Dittié et al.,
1997). Since both the tyrosine motif and the acidic cluster are
also present in M6PRs we have investigated whether or not AP1 recruitment onto ISGs in PC12 cells involves interaction with
M6PRs. We have studied the distribution of the M6PRs in ISGs
using subcellular fractionation and immunogold labelling of
cryosections. We show here that the cytoplasmic domain of the
CI-M6PRs can interact with AP-1 in a CKII phosphorylationdependent fashion and that M6PRs are also responsible for
recruiting AP-1 to ISGs. In addition, using sequential
immunoisolation techniques we have demonstrated that at most
50% of ISGs contain the CI-M6PR, while furin in contrast is
present in greater than 80% of ISGs. Together these data
establish that ISGs from PC12 cells exhibit a differential
distribution of the M6PRs and furin, and both M6PRs as well
as furin can recruit AP-1 to ISGs.
MATERIALS AND METHODS
Reagents
Carrier-free [35S]sulfate and 125I-Protein A were from AmershamPharmacia Biotech, UK. Nucleotides, creatine phosphate, and creatine
phosphokinase were from Boehringer Mannheim, Germany. Casein
kinase II was prepared according to the method of Litchfield et al.
(1990). Heparin and horseradish peroxidase (HRP type II) were from
Sigma, UK. Tautomycin and microcystin LR were obtained from
Calbiochem, UK. Fine chemicals were from Merck, UK, Life
Technologies, UK and Sigma, UK.
Cells and antibodies
PC12 cells, a rat pheochromocytoma cell line (clone 251; Heumann et
al., 1983), originally obtained from Dr H. Thoenen (Martinsried,
Germany), were maintained as described (Tooze and Huttner, 1992).
The mAb against bovine γ-adaptin (100/3) (Sigma) was used at a
dilution of 1:200. Polyclonal antibodies against furin (van Duijnhoven
et al., 1992 and Dittié et al., 1997), and the cytoplasmic domain of the
CI-M6PR (STO-49 and 52, prepared using the GST-fusion proteins,
see below) were used at dilutions of 1:500-1:1000. Anti-SgII antibody
(175) was as previously described (Dittié and Tooze, 1995). The antication dependent mannose-6-phosphate receptor (CD-M6PR)
antibody, used at 1:1000 or as previously described (Klumperman et
al., 1993), was a kind gift from Dr A. Hille-Rehfeld, Göttingen,
Germany. Secondary antibodies were HRP-conjugated sheep antimouse or sheep anti-rabbit (Amersham-Pharmacia Biotech, UK).
GST-fusion proteins and expression constructs
Different regions of the cytoplasmic domain of the mouse CI-M6PR
(Dittié et al., 1997, see Fig. 5A) were cloned via RT-PCR from NIH3T3 cell mRNA and ligated in frame to the COOH terminus of GST
using the pGEX-3X system (Amersham-Pharmacia Biotech, UK).
The fusion proteins were expressed in BL21 cells and purified using
glutathione Sepharose 4B (Amersham-Pharmacia Biotech, UK). The
fusion proteins were also used to generate polyclonal antibodies
(STO-49 and STO-52).
The full-length sequence of human furin preceded by the flag tag
(Kodak, UK) was subcloned from hfur/flag pGEM7Zft (provided by
Dr Gary Thomas, Oregon, USA) into pBK/CMV (Stratagene).
Transient transfection was carried out using electroporation (Bio-Rad,
UK) and 1×106 cells. Six hours after transfection 5 mM Na butyrate
was added to the cultures and they were incubated for an additional
18 hours before being processed for cryomicroscopy.
Preparation of PC12 TGN, ISGs, MSGs and bovine brain
cytosol
PC12 cells were pulse-labelled with [35S]sulfate and chased at 37°C
(Tooze and Huttner, 1990; see figure legends for details). ISGs,
constitutive secretory vesicle (CSVs), MSGs, and TGN were prepared
from either labelled or unlabelled PC12 cells after preparation of a
post nuclear supernatant (PNS) by velocity and equilibrium sucrose
gradient centrifugation (Dittié et al., 1996). TGN was enriched in
velocity gradient fraction 9, CSVs were found in equilibrium gradient
(EG) fractions 5 and 6, ISGs in EG fractions 7-9, and MSGs in EG
fractions 10-12. The ISGs used for the binding assay contained ~0.5
mg/ml protein.
Equal volumes of each fraction (100 µl from the velocity gradient
fractions and 200 µl from the equilibrium gradient fractions) were
used for western-blot analysis and the bound antibodies were
visualized by ECL (Amersham-Pharmacia Biotech, UK). Bovine
brain cytosol was prepared as described (Dittié et al., 1996). Protein
concentrations were determined by protein assay (Bio-Rad, UK) using
IgG as a standard.
Horseradish peroxidase uptake
To determine the position of early and late endosomes after sucrose
M6PRs are found in a subset of PC12 cell ISGs 3957
gradient centrifugation, PC12 cells were incubated with the
endocytic fluid-phase marker HRP. Two 15 cm dishes of
subconfluent PC12 cells each were washed twice in DMEM/0.1%
HS/0.05% FCS (low serum DMEM) and incubated with 2 mg/ml
HRP in low serum DMEM for 7 minutes at 37°C either without
chase to label early endosomes or followed by a 30 minute chase
with normal growth medium to label late endosomes. A PNS was
prepared from each and subjected to velocity and equilibrium
centrifugation. The HRP activity in each fraction was determined
using o-dianisidine (Marsh et al., 1987).
Immunoisolation
Immunoisolation of [35S]sulfate labelled ISGs using polyclonal
antibodies against CI-M6PR and furin was carried out as described
(Dittié et al., 1996), using 100 µl of the ISG containing equilibrium
gradient fractions 8 and 9. When two rounds of isolation were
performed, the supernatant of the incubation with the first round
antibody was transferred to a fresh tube and incubated for the same
time with the second round antibody. For competition experiments 0.1
mg/ml GST-CI-M6PR or GST-furin fusion proteins were added before
the immunoisolation and were also included at the same
concentrations throughout the immunoisolation. The final supernatant
was TCA precipitated and resuspended in SDS sample buffer for
PAGE analysis.
RESULTS
Localization of the CI- and CD-M6PRs by subcellular
fractionation of PC12 cells
Using immunogold labelling techniques M6PRs have been
shown to be present in ISGs and to co-localize with AP-1 in
both rat endocrine pancreatic β-cells and exocrine parotid and
pancreatic cells (Klumperman et al., 1998). To demonstrate
that the M6PRs are also present in ISGs of PC12 cells we
fractionated PC12 cells (Dittié et al., 1996) and probed each
fraction with antibodies specific for either the CI- or CDM6PR. The CI-M6PR antibody was raised against a GSTfusion protein encoding most of the cytoplasmic domain of the
mouse CI-M6PR and was characterized by comparison with a
previously published antibody raised against the CI-M6PR
(Brown et al., 1995; data not shown). Using these antibodies
we found that the distribution of both the CI-M6PR and CDM6PR in PC12 cell membranes after fractionation of a postnuclear supernatant using a velocity controlled sucrose
gradient was complex (Fig. 1A). The CI-M6PR was found in
Cell-free assay to reconstitute γ-adaptin binding to PC12
ISGs
As previously described in detail (Dittié et al., 1996), ISGs were
incubated with varying amounts of bovine brain cytosol in 25 mM
Hepes, pH 7.2, 25 mM KCl, 2.5 mM MgOAc (binding buffer) and
100 µM GTPγS. After incubation for 30 minutes at 37°C the reaction
was stopped, the ISGs were sedimented and the γ-adaptin bound was
quantitated after western blotting using 125I-Protein A.
Competition experiments using GST-fusion proteins were
performed as follows: 0.5 mg/ml bovine brain cytosol was
preincubated for 15 minutes at 4°C in presence of 0.1 µM tautomycin
and 1 µM microcystin LR with GST-fusion proteins (5 µM) either
non-phosphorylated or phosphorylated by purified CKII as described
by Dittié et al. (1997). This reaction mixture was then used for a
standard binding assay.
Binding of adaptor components to immobilized furin tails
Fusion proteins were purified from BL21 cells expressing the
different GST-CI-M6PR constructs (see Fig. 5A). The purified
fusion proteins were dialyzed against 50 mM Tris-HCl, pH 7.4, 140
mM KCl, 10 mM MgCl2 and incubated with or without GST-CKIIα
(Dittié et al., 1997) in the presence of ATP and regenerating system
for 1 hour at 37°C, then stopped by the addition of heparin. 10 µg
of phosphorylated fusion protein were coupled to 30 µl of
glutathione Sepharose 4B (50% w/v) for 4 hours at 4°C. Binding of
AP-1 to the immobilized fusion proteins was performed as described
before (Dittié et al., 1997).
Electron microscopy of ultrathin cryosections
PC12 cells were prepared for ultrathin cryosectioning and doubleimmunogold labeling according to the Protein A-gold method as
previously described (Slot et al., 1991). In short, cells were fixed in a
mixture of 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M
phosphate buffer for 2 hours at room temperature and stored overnight
at 4°C in 2% paraformaldehyde. The cells were then washed in
PBS/0.2 M glycine, scraped off the dish and pelleted in 10% gelatin
in PBS, which was solidified on ice and cut into small blocks. After
overnight infiltration with 2.3 M sucrose at 4°C, blocks were picked
up in a 1:1 mixture of sucrose and methyl cellulose (Liou et al., 1996).
PC12 cells overexpressing furin were incubated overnight with 5 mM
butyrate prior to fixation. Sections were viewed in a JEOL 1010
transmission electron microscope operating at 80.0 kV.
Fig. 1. Subcellular fractionation reveals that both the CI- and the CDM6PR are found in fractions which contain ISGs but not MSGs.
(A) Fractions from a velocity sucrose gradient of a post-nuclear
supernatant obtained from PC12 cells were immunoblotted with
antibodies to the CI- and CD-M6PRs. The location of the light and
heavy vesicle population, and the TGN is shown. (B) The light or (C)
heavy vesicle pool was subjected to equilibrium sucrose
centrifugation. The location of the CSV (constitutive secretory
vesicle), ISGs and MSGs are shown. Fractions from each gradient
were analysed by immunoblotting with antibodies to the CI- and CDM6PRs. We have reproducibly detected a strong signal for CDM6PR in the fractions analysed in B although there is apparently
very little immunoreactivity in the starting pool (fractions 2-4 in A).
The location of the fractions enriched in ISGs and MSGs on the light
and heavy vesicle pool gradients is illustrated. The position of the
enriched fractions containing ISGs and MSGs was confirmed by
analysis of the marker protein SgII (not shown). For all gradients
fractions were collected from the top of the gradient (fraction 1 top).
3958 A. S. Dittié, J. Klumperman and S. A. Tooze
Location of endosomal compartments in PC12 cells
by subcellular fractionation
As both M6PRs are present in early and late endosomes (for
review see Hille-Rehfeld, 1995) it was important to determine
what precentage of the immunoreactivity specific for the CIand CD-M6PRs on the velocity and equilibrium sucrose
gradients is present in endosomal compartments. To identify
endosomes we incubated PC12 cells with horseradish
peroxidase (HRP) for different periods of time. The cells were
then homogenized and fractionated as in Fig. 1, and the HRP
activity was assayed for across the gradients. After HRP is
internalized for 7 minutes to label early endosomes, the bulk
of the early endosomes were present in fraction 5-7 of the
velocity controlled sucrose gradient (Fig. 2A). These fractions
(5-7) from the velocity gradients, which contain the bulk of the
early endosomes were pooled and subjected to equilibrium
sucrose gradient centrifugation. The distribution of the HRP
activity across the equilibrium gradient demonstrated that the
early endosomes were found in fractions 5-8, with a peak in
fraction 7 (Fig. 2C). The light vesicle pool (fractions 2-4) of
the velocity gradient, which is equivalent to the light vesicle
pool in Fig. 1, contained a low level of HRP activity (Fig. 2B).
We estimate that only about 20% of all early endosomes are
found in the light vesicle fraction from the velocity sucrose
gradient. Interestingly, after equilibrium sucrose gradient
centrifugation of the light pool the HRP activity was in
fractions 5-8 of the equilibrium gradient and exhibited a very
similar distribution to the HRP activity found in the pool of
velocity gradient fractions 5-7 after equilibrium sucrose
gradient centrifugation (compare Fig. 2B and C). This result
suggests that the early endosomal compartment is
heterogeneous with respect to shape and size as it is widely
spread across the velocity gradient but has a uniform density
as it sediments to a defined position on the equilibrium
A) VG: PNS from PC12 cells
7min HRP pulse/no chase
7min HRP pulse/30min chase
HRP activity
(absorbance at OD44 5 mn)
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
8
9 10 11 12
B) EG: pool of fractions 2 - 4
HRP activity
(absorbance at OD44 5 mn)
0.15
0.125
0.1
0.075
0.05
0.025
0
1
2
3
4
5
6
7
8
7
8
9 10 11 12
C) EG: pool of fractions 5 - 7
0.2
HRP activity
(absorbance at OD44 5 mn)
most fractions, with a peak of immunoreactivity in fractions 59, while the bulk of the CD-M6PR immunoreactivity was
found in fractions 5-9.
To increase the resolution of the different compartments
present in the velocity gradient fractions, a light and a heavy
vesicle pool was prepared containing ISGs and MSGs,
respectively, and subjected to equilibrium sucrose gradient
centrifugation. ISGs which are present in fractions 2-4 of the
velocity gradient, sediment to fractions 7-9 on the equilibrium
gradient. MSGs, which are present in fractions 4-7 of the
velocity gradient, sediment to a denser sucrose concentration
and are found in fractions 9-12 on the equilibrium gradient,
with the majority in fractions 11 and 12 (Tooze et al., 1991).
After fractionation of the light vesicle pool (fractions 2-4) on
equilibrium gradients immunoblotting revealed that both the
CI-and CD-M6PR were found in fractions 6-9 (Fig. 1B).
Likewise, after fractionation of the heavy vesicle pool
(fractions 4-7) on equilibrium gradients the immunoreactive
peaks of both the CI- and the CD-M6PR were also found in
fractions 6-9 (Fig. 1C). These results demonstrate three points:
first, and as expected, the CI-M6PR and the CD-M6PR have
similiar fractionation profiles on the gradients described above;
secondly, some of both the CI- and the CD-M6PR is present
in the fractions containing the ISGs; lastly, neither the CI- nor
CD-M6PR co-localized on equilibrium gradients with MSGs
despite co-localization on the velocity gradient.
0.15
0.1
0.05
0
1
2
3
4
5
6
9
10 11 12
fraction No.
Fig. 2. Analysis of the position of endosomal fractions, using HRP
activity after internalization for 7 minutes or 7 minutes followed by a
30 minute chase. (A) HRP activity across the velocity sucrose
gradient for a PNS from PC12 cells loaded with HRP for 7 minutes
or 7 minutes followed by a 30 minute chase. The position of the
fractions loaded onto the subsequent equilibrium gradients are
shown. (B) Equilibrium sucrose gradient centrifugation of the pool of
fractions 2-4 from the velocity sucrose gradient loaded with a PNS of
PC12 cells after a 7 minute internalization of HRP. (C) Equilibrium
sucrose gradient centrifugation of the pool of fractions 5-7 from the
velocity sucrose gradient loaded with a PNS of PC12 cells after a 7
minute internalization of HRP.
gradients. From these results it is possible that some of the CIand CD-M6PR immunoreactivity in the fractions enriched for
ISGs is present in contaminating early endosomes. However,
some CI- and CD-M6PRs are probably also present in ISGs
because the peaks of immunoreactivity (Fig. 1B) and HRP
activity (Fig. 2B) are not absolutely coincident. A 7 minute
internalization of HRP followed by a 30 minute chase revealed
M6PRs are found in a subset of PC12 cell ISGs 3959
that the late endosomes and lysosomes rapidly sedimented into
the bottom half of the velocity gradient (fractions 6-12 with a
peak in fraction 9) well separated from the post-Golgi vesicle
pool in fractions 2-4 (data not shown).
Antibodies to the CI-M6PR can immunoisolate
immature secretory granules
To determine more precisely what percentage of ISGs have
M6PRs, we performed an immunoisolation experiment using
fractions 8 and 9 from the equilibrium gradient which are
enriched in ISGs. The ISG, isolated from cells labelled with
[35S]sulfate for 5 minutes followed by a 15 minute chase,
contained [35S]sulfate labelled SgII. After incubation with an
anti-CI-M6PR antibody approximately 30% of the total ISGs
containing [35S]sulfate labelled SgII could be immunoisolated
(Fig. 3A, lane 1, and Table 1). As Fig. 3A, lane 3, and Table 1
show this signal could be competed by pre-incubation of the
antibody with the GST-fusion protein containing the
cytoplasmic domain of the CI-M6PR (GST-CT87, see Fig. 5).
Although sulfate-labelled ISGs were immunoisolated with the
anti-CI-M6PR antibody the efficiency of immunoisolation was
not very high: over the course of several experiments we
observed that a maximum of 45% of the total sulfate-labelled
ISGs was immunoisolated with the anti-CI-M6PR antibody
(Table 1).
The results obtained with the anti-CI-M6PR antibody were
in contrast with those previously obtained using an anti-furin
antibody with which, in similar immunoisolation experiments,
greater than 85% of ISGs could be immunoisolated (Table 1
and Dittié et al., 1997). It was possible that the anti-CI-M6PR
antibody did not recognize the cytoplasmic domain of the CIM6PR as efficiently as the cytoplasmic domain of furin was
recognized by the anti-furin antibody. To determine if the antiM6PR antibody was efficiently binding the cytoplasmic
domain of the M6PR in membranes we performed an
immunoisolation experiment with an enriched fraction of TGN
membranes, obtained from cells labelled with a 5 minutes
pulse of sulfate. We found that the anti-M6PR receptor
antibody was able to immunoisolate 84% of the total
[35S]sulfate present in the TGN fraction. In addition, the GSTCT87 fusion protein was able to compete the antibody binding
to background levels (data not shown). These results suggest
that the anti-M6PR antibody is able to recognize the
cytoplasmic domain of the M6PR under the conditions used
Fig. 3. Immunoisolation of ISGs containing [35S]sulfate labelled SgII with antibodies directed against the cytoplasmic domain of CI-M6PR and
furin. (A) ISGs were obtained from PC12 cells pulse-labelled for 5 minutes and chased for 15 minutes following velocity and equilibrium
sucrose gradient centrifugation. ISGs (total sample lane 5) were incubated with an antibody to the CI-M6PR (lanes 1 and 2) or anti-CI-M6PR
antibody preincubated with the GST-fusion protein used to immunize rabbits (lane 3 and 4). The material immunoisolated bound to the Staph A
(P) was solubilized in SDS-sample buffer while the unbound (S) was TCA precipitated and solubilized in SDS-sample buffer. (B) Sequential
immunoisolation was performed on the ISG fraction by incubation first (P1) with anti-CI-M6PR antibody (lanes 1, 4 and 7) or anti-furin
antibody (lane 10 and 12). The unbound supernatant was then collected and subjected to a second round of immunoisolation (P2) using preimmune sera (lane 2), anti-CI-M6PR antibody (lanes 5 and 13), or anti-furin antibody (lane 8) or analysed directly as above. Positions of SgII
and chromogranin B (CgB) are indicated.
3960 A. S. Dittié, J. Klumperman and S. A. Tooze
Table 1. Quantitation of immunoisolation of ISGs with
anti-M6PR and anti-furin antibodies
Single round of immunoisolation:
Antisera
Pre-immune
Anti-M6PR
Anti-M6PR +
GST-M6PR
Anti-furin
No.
of exp.
% total
in pellet
% total in
supernatant
4
5
5
8.3±6.7
29±4.4
1.8±2.4
92±6.7
72±4.4
98±2.4
3
85±1.0
15±1.0
Sequential round of immunoisolation:
No. of
Exp.
% total in
1st pellet
% total in
2nd pellet
% total in
supernatant
1 rd. anti-M6PR
2 rd. pre-immune
2
45
8
47
1 rd. anti-M6PR
2 rd. anti-M6PR
2
43
5.6
51
1 rd. anti-M6PR
2 rd. anti-furin
2
37
45
19
1 rd. anti-furin
2 rd. anti-M6PR
3
99±2.3
1.3±2.3
0
Antisera
Either a single or sequential round of immunoisolation was performed with
the indicated antibodies. For single immunoisolation experiments the antigenantibody complex was recovered with Staph A and is referred to as the pellet,
while the unbound material was found in the supernatant. For sequential
immunoisolation experiments the unbound supernatant from the first round
was collected and used as the starting material for the 2nd round of
immunoisolation. The percentages shown are derived from a total obtained by
addition of the 35S radioactivity present in SgII found in the pellets and
supernatants and the errors are expressed as standard deviation.
for immunoisolation, and does so as efficiently as the anti-furin
antibody.
An alternative explanation for the low immunoisolation
efficiency of the ISGs with the anti-M6PR antibody is that the
ISGs are a heterogeneous population with a variable amount
of the CI-M6PR and furin in their membrane. ISGs which do
not have sufficient amounts of the CI-M6PR would not be
immunoisolated with the anti-CI MPR antibody, but would be
efficiently immunoisolated with the anti-furin antibody if
sufficient amounts of furin was present. To address this we
performed a sequential immunoisolation experiment where the
ISGs were incubated with the anti-CI-M6PR antibody, the
antibody-antigen complexes were immobilized on Staph A and
the supernatant was supplemented with the anti-furin antibody,
or, in reverse order, the anti-furin antibody was followed by the
anti-CI-M6PR antibody (Fig. 3B). As in Fig. 3A, incubation
with the anti-CI-M6PR antibody resulted in approximately 3045% of the labelled ISGs (Fig. 3B, lanes 1, 4 and 7, and Table
1) binding to the Staph A. A subsequent readdition of the antiCI-M6PR antibody to the unbound fraction resulted in no
further recovery of labelled ISGs (Fig. 3B, lane 5, and Table
1) compared to pre-immune serum (Fig. 3B, lane 2, and Table
1). However, addition of the anti-furin antibody to the unbound
supernatant fraction resulted in the recovery of virtually all the
remaining sulfate labelled ISGs (Fig. 3B, lane 8, and Table 1).
To control for the efficiency of the sequential
immunoisolation experiment we performed additional
experiments
with
the
anti-furin
antibody.
After
immunoisolation with the anti-furin antibody in which greater
than 85% of the ISGs (Fig. 3B, lane 10, and Table 1) were
Fig. 4. Ultrathin cryosections showing Golgi (G) regions of wildtype (A,B) and furin overexpressing (C-E) PC12 cells, illustrating the
presence of CD-M6PR (B) and furin (C-E) in secretory granules (S).
(A) Immunogold labelling of SgII identified ~150 nm diameter
organelles with a partially condensed content as secretory granules.
Note that a dense staining on the cytoplasmic side of the membrane
(arrows in A, B, C, D, and E), indicative for the presence of clathrin,
covered large areas of some secretory granules, thereby defining
them as ISGs. (B) SgII: 10 nm gold; CD-M6PR: 15 nm gold. The
closed arrowheads point to CD-M6PR staining in SgII positive
secretory granules (S). Also CD-M6PR negative secretory granules
(open arrowhead) were regularly observed in the Golgi area. (C and
D) SgII: 10 nm gold; furin 15 nm gold. Furin is localized in a SgIIpositive structures similar to a condensing vacuole (arrows) and
secretory granules (S), some of which are clathrin coated (arrows).
E) Furin (10 nm gold) and CD-M6PR (15 nm gold) sometimes colocalized to the same TGN vesicle (arrowheads), some of which were
clathrin coated (upper vesicle). L, lysosome. Bars, 200 nm.
immunoisolated, addition of the anti-CI-M6PR antibody to the
supernatant failed to recover any additional labelled ISGs (Fig.
3B, lane 13, and Table 1). The signal obtained with the antifurin and anti-CI-M6PR antibodies used either in the first and
second rounds could be competed by the addition of the
respective GST-fusion proteins used to raise the antisera (see
Fig. 3A and Dittié et al., 1997). These results demonstrate that
while only 45% at most of the sulfate labelled ISGs could be
immunoisolated with anti-CI-M6PR antibodies, most (greater
than 85%) could be immunoisolated with the anti-furin
antibody. Furthermore, the ISGs which were not bound to the
Staph A via the anti-CI-M6PR antibodies could be
immunoisolated with the anti-furin antibody. This suggests that
while furin is present on virtually all ISGs, at least half of the
ISGs do not have sufficient amounts of CI-M6PR to allow them
to be immunoisolated. We have not investigated the
distribution of the CD-M6PR because the anti-CD-M6PR
antibody did not efficiently immunoisolate any membranes
(even control TGN membranes) under the conditions used for
immunoisolation.
Finally, as an additional control for the efficiency of the
immunoisolation experiments we used the same anti-CI-M6PR
antisera to immunoisolate early endosomes, labelled after a 5
minute HRP internalisation, prepared from velocity gradients
(pool of fractions 6 and 7). Early endosomes could be
immunoisolated with both the anti-furin antibodies and antiCI-M6PR antibodies (data not shown). Under conditions where
quantative immunoisolation is obtained, comparison of the
recovery of early endosomes revealed that with the anti-furin
antibody approximately 2-fold more early endosomes were
isolated than with the anti-CI-M6PR antibody (data not
shown). Likewise with the anti-furin antibodies we were able
to immunoisolate approximately 2-fold more ISGs than with
the anti-CI-M6PR antibodies (see Fig. 3). These results imply
that in PC12 cells furin is more abundant than the CI-M6PR
in both early endosomes and ISGs.
Localization of the CD-M6PR to immature secretory
granules
To confirm the co-localization of the M6PR and furin on ISGs
we performed immunogold labelling of cryosections of PC12
cells (Fig. 4). Furin is present in very low amounts in cells
(Creemers et al., 1993) and not readily detectable by
M6PRs are found in a subset of PC12 cell ISGs 3961
Fig. 4
3962 A. S. Dittié, J. Klumperman and S. A. Tooze
immunogold labelling techniques (J. Klumperman,
unpublished results) so to perform this analysis it was
necessary to transiently transfect PC12 cells with furin. To
unequivocally define ISGs in the Golgi area, we first localized
SgII, a secretory granule marker protein, to organelles with a
partially condensed dense core content (Fig. 4A). Large areas
of some of these ISGs were coated with clathrin, identified by
its characteristic appearance, consistent with our previous
localization of γ-adaptin on isolated ISGs. Previous
immunogold labelling studies (Klumperman et al., 1993) have
shown that CI-M6PR and CD-M6PR co-localize to the same
TGN membranes in HepG2 cells. We used anti-CD-M6PR
antibodies to study the subcellular distribution of M6PRs in
situ in PC12 cells. Double immunogold labelling of SgII and
CD-M6PR (Fig. 4B) or SgII and furin (Fig. 4C and D) showed
the presence of both CD-M6PR and furin in ISGs, some of
which clearly exhibited a clathrin coat. Additional labelling in
the Golgi area was found on TGN membranes; the CD-M6PR
and furin were co-localized in clathrin-coated and in nonclathrin coated vesicles (Fig. 4E) that are likely to mediate
transport between TGN and endosomes.
These results demonstrate that the CI-M6PR and CDM6PR are found in ISGs along with furin, and some of these
ISG structures are coated with clathrin. Interestingly, our data
indicate that not all ISGs contain the CI-M6PR. As we have
previously demonstrated that furin is involved in AP-1
recruitment to ISGs (Dittié et al., 1997) and others have
shown that M6PRs are key components for recruitment of
AP-1 to the TGN (for review see Le Borgne and Hoflack,
A)
1998) we were interested in role the M6PR plays in coat
recruitment on ISGs.
Preparation and phosphorylation of GST-CI-M6PR
fusion proteins
To determine if M6PRs are involved in clathrin coat recruitment
to ISGs we used the established in vitro assay (Dittié et al.,
1996) and fusion proteins containing the cytoplasmic domain
of the CI-M6PR to compete for AP-1 binding. To develop a
reagent containing the cytoplasmic domain of the CI-M6PR
which could bind to AP-1 in cytosol we constructed a series of
GST-fusion proteins containing different lengths of the
cytoplasmic domain of the mouse CI-M6PR (Fig. 5A). The CIM6PR cytoplasmic sequence has two stretches of acidic amino
acids which are sites for CKII phosphorylation in vivo and in
vitro, as well as a tyrosine-based sorting motif and a di-leucine
sorting motif. We chose to express fusion proteins containing
only the sites which interact with AP-1, that is the two CKII
sites and the di-leucine motif. Fusion proteins were prepared
which contained all three sites (GST- CT87), just the two CKII
sites (GST-∆4), only the N-terminal CKII site (GST-∆9), or the
C-terminal CKII site and the di-leucine site (GST-CT13).
Bacteria expressing these constructs produced fusion proteins
of the correct molecular mass which were purified on
glutathione-Sepharose (Fig. 5B). Expression of the GST-CT87
construct in bacteria gave rise to three bands, the full length
fusion protein having the lowest mobility by SDS-PAGE (Fig.
5B, lane 3) while the other constructs produced a single band
of the correct molecular mass (Fig. 5B).
1
CI-M6PR (mouse):
2315 2344
2294
35
Y
SP
N
luminal
2399
2474
S
TM
GST
N
GST
GST CT87 (aa2395-2482):
N
GST ∆4 (aa2395-2478):
N
Fig. 5. Construction and expression of truncated
cytoplasmic domains of mouse CI-M6PR as GST
fusion proteins. (A) Scheme of constructs. Various
sections of the C-terminal region of the cytoplasmic
domain of the mouse CI-M6PR were fused to GST to
create the GST-fusion proteins illustrated. (B) These
GST-fusion proteins were expressed and purified on
glutathione Sepharose as detailed in Materials and
Methods and analysed by Coomassie blue staining.
Except for CT87, the major band of all constructs
expressed was the correct size fusion protein. The full
length fusion protein for CT87 was the slowest
migrating band at 44 kDa which corresponds to the
predicted molecular mass. (C) The purified GST
fusion proteins were incubated with recombinant
CKIIα to test their ability to be phosphorylated. The
full length fusion proteins, except for GST, were in
each case phosphorylated by CKIIα. The arrowhead
indicates the CKIIα fusion protein which is
autophosphorylated.
GST ∆9 (aa2395-2473):
N
2395 2399
S
GST
2395 2399
S
GST
C
cytoplasmic
GST CT13 (aa2469-2482):
2395 2399
S
2482
S LL
2469
2482
SLL C
2474
2482
S LL C
2474 2478
S C
2473
C
M6PRs are found in a subset of PC12 cell ISGs 3963
As AP-1 binding to the cytoplasmic domain of the CI-M6PR
depends on CKII phosphorylation of the receptor (Le Borgne
et al., 1993), we next tested if the GST-CI-M6PR fusion
proteins could be phosphorylated by CKII by incubating the
fusion proteins with recombinant CKIIα and [32P]ATP (Fig.
5C). The full length form of all four GST-fusion proteins but
not GST itself could be phosphorylated by recombinant CKIIα.
The increased sensitivity obtained using 32P revealed that the
all the GST-fusion protein preparations contained low amounts
of truncated fusion proteins but the full-length protein was the
major form.
Fig. 6. Optimal AP-1 binding to CI-M6PR in vitro requires CKII
phosphorylation of Ser2474. (A) GST or GST-fusion proteins bound
to glutathione Sepharose were incubated with or without
recombinant CKIIα at 37°C for 60 minutes, then incubated in bovine
brain cytosol for an additional 30 minutes at 37°C. AP-1 bound to
beads was assayed by immunoblotting with mab100/3.
(B) Phosphorylation dependent stimulation of AP-1 binding to GSTfusion proteins is expressed relative to the unphosphorylated GSTfusion proteins. Values are obtained from 7 independent experiments
for GST and GST-CT87 and 4 independent experiments for GSTCT13, ∆4 and ∆9. (C) GST and GST-fusion proteins furin and GSTCT87 were preincubated with or without purified CKIIα as above.
Bovine brain cytosol was preincubated with the GST and the GSTfusion proteins for 15 minutes before incubation with ISGs in the
binding assay. The reduction in the amount of bovine AP-1 recruited
to the ISGs is expressed relative to the control, unphosphorylated
GST. The error bars represent standard deviation of the mean.
CKII phosphorylated CI-M6PR cytoplasmic domains
can bind AP-1 and compete AP-1 recruitment to
ISGs
The GST-M6PR fusion proteins containing variable lengths of
the cytoplasmic tail of CI-M6PR (see Fig. 5A) were
phosphorylated with CKII, immobilized to glutathioneSepharose and then incubated with bovine brain cytosol. AP1 binding was then assayed by immunoblotting with the bovine
specific γ-adaptin mAb 100/3. As shown in Fig. 6A, the
immobilized fusion proteins GST-CT13, GST-CT87, and GST∆4 could efficiently bind AP-1 after phosphorylation by CKII.
These constructs all have the C-terminal CKII site consisting
of Ser2474. Interestingly, neither the unphosphorylated fusion
proteins, nor the phosphorylated GST-∆9, which contains only
the N-terminal CKII site close to the transmembrane region,
bound AP-1. Although there was some binding of AP-1 to GST
with and without incubation with CKII, there was a significant
phosphorylation-dependent stimulation of AP-1 binding for
GST-CT87, as well as slight stimulation for GST-CT13 and
GST-∆4 (Fig. 6B). These results suggest that the CKII site at
the C-terminal end of the cytoplasmic domain of CI-M6PR,
and in particular Ser2474 is required for the efficient binding of
AP-1.
To determine if the CI-M6PR was involved in recruitment
of AP-1 to ISGs from PC12 cells we performed a competition
experiment using the cell-free binding assay (Dittié et al.,
1996). This assay measures ARF1-dependent recruitment of
exogenous bovine AP-1, present in bovine brain cytosol, onto
ISG membranes. Bovine brain cytosol was preincubated with
the phosphorylated or non-phosphorylated GST-CT87 fusion
protein, and then the entire mixture was added to the assay
containing ISGs in the presence of GTPγS (Fig. 6C).
Recruitment of exogenous AP-1 was then assayed with mAb
100/3 (Ahle et al., 1988). As controls we used GST or a GSTfurin fusion protein, previously characterized (Dittié et al.,
1997), either with or without prior incubation with CKII. GST
and GST after incubation with CKII were unable to compete
for AP-1 binding to ISGs. The phosphorylated GST-furin
fusion protein which has been previously shown to compete for
binding of AP-1 to ISGs (Dittié et al., 1997) inhibited binding
about 50%, while the phosphorylated cytoplasmic domain of
the CI-M6PR (GST-CT87) was effective in reducing binding
by about 70%. Both the unphosphorylated GST-furin and GSTM6PR fusion proteins did not significantly inhibit binding.
These results demonstrate that after CKII phosphorylation the
CI-M6PR cytoplasmic domain can effectively compete for AP1 recruitment to ISGs and reduces AP-1 binding below that
observed with phosphorylated furin fusion protein.
DISCUSSION
There is a growing body of evidence that in both endocrine and
exocrine cells, proteins are removed from the regulated
secretory pathway by vesicles which originate from maturing
secretory granules. Some soluble proteins, for example Cpeptide which is derived from proinsulin in endocrine
pancreatic cells (Arvan et al., 1991), are removed from the ISG
and secreted by constitutive-like secretion (for review see
Arvan and Castle, 1998) into the extracellular space, while
other soluble molecules, such as lysosomal enzymes, are
3964 A. S. Dittié, J. Klumperman and S. A. Tooze
transported from the ISG to the endosome. While the transport
of lysosomal enzymes from the ISG to the endosome depends
on M6PRs (Kuliawat et al., 1997), it is not clear how C-peptide
is removed from the ISG, and if it occupies the same vesicles
as the M6PR and lysosomal enzymes. Furthermore, the
vesicular carrier for other molecules such as furin, which is
found in ISGs but not MSGs, is not known although furin in
the ISG can bind AP-1 (Dittié et al., 1997). These results
suggest either that vesicles budding from the ISG carry a
variety of cargo molecules or that there is more than one exit
route from the ISG. The existence of multiple exit routes from
the ISG is supported by data suggesting that there is a recycling
pathway from the ISG back to the TGN, distinct from the
constitutive-like secretory vesicles, to retrieve molecules such
as the membrane form of peptidylglycine α-amidating
monooxygenase (Milgram et al., 1994).
To gain further insight into the process of vesicular transport
from the ISG during secretory granule maturation, we have
focused on trying to understand the role of AP-1 containing
clathrin-coats in this process. Previous results using
immunogold labelling suggest that the M6PR (Klumperman et
al., 1998) uses the AP-1 coat-formation machinery to be
removed from ISGS by CCVs. Sequences in the cytoplasmic
domain of M6PRs that are implicated in AP-1 binding to TGN
membranes, as well as the TGN localization of M6PRs, are the
di-leucine motif, the tyrosine motif, and an acidic cluster of
amino acids constituting a CKII phosphorylation site, which
when phosphorylated may be the most important for high
affinity binding of AP-1 (for review see Le Borgne and
Hoflack, 1998). Similar motifs in furin are likewise thought to
bind AP-1 in TGN membranes (Jones et al., 1995; Schäfer et
al., 1995; Takahashi et al., 1995). As phosphorylation of the
acidic cluster of amino acids in furin is an important signal for
recruitment of AP-1 to ISGs (Dittié et al., 1997), and since
furin is absent from MSGs, one can assume that furin is
removed by AP-1 containing CCVs from the ISGs.
While the CCVs containing the M6PR most probably go to
the endosome, it is more difficult to make predictions about the
final destination of the furin containing CCVs. One recent
model suggests that the phosphorylated CKII site modulates
retrieval of furin from the ISG to the TGN (Molloy et al., 1999)
which raises several additional questions about the
mechanisms of transport of other proteins out of the ISG, and
in particular the M6PR and lysosomal enzymes. Does the
M6PR recently shown to be present on ISGs and co-localized
with AP-1 (Klumperman et al., 1998) also use the CKII
phosphorylated acidic clusters in the cytoplasmic domain to
bind AP-1 in the ISGs? Are furin and the M6PR found in the
same AP-1 containing clathrin coated vesicles? and finally
does the M6PR go back to the TGN from the ISG or does it
go directly to an endosomal compartment? The recycling of
M6PRs to the TGN seems most unlikely in view of recent data
concerning the co-localization of syntaxin 6 and the CD-M6PR
in cells with a regulated secretory pathway (Klumperman et al.,
1998) and raises the possibilities that either furin and the
M6PR are in different vesicles or that furin is also targeted to
endosomes from the ISG and not to the TGN.
As reported here the CI-M6PR cytoplasmic domain can
recruit AP-1 from cytosol and this recruitment requires prior
phosphorylation by CKII. AP-1 binding to ISGs can be
inhibited by preincubation of the cytosol with the
phosphorylated GST-CI-M6PR fusion protein containing both
acidic clusters, the sites for CKII phosphorylation, and the dileucine motif. The GST-fusion proteins GST-CT13 and GST∆4 bind AP-1 better than GST-∆9 (Fig. 6A), which does not
contain the C-terminal CKII site, implying that the
phosphorylation of the acidic clusters, and in particular the Cterminal phosphorylated Ser2474 residue, is the most important
signal for AP-1 binding. Furthermore, deletion of the dileucine motif resulted in a decrease in the amount of AP-1
bound in a manner which was stimulated by phosphorylation
(see Fig. 6B) but did not affect the total amount bound (see
Fig. 6A). Others (Johnson and Kornfeld, 1992; Mauxion et al.,
1996) have reported that AP-1 binding requires the presence
of di-leucine motifs and Chen et al. (1997) have demonstrated
that di-leucine motifs are required for sorting of cathespin D
by the CI-M6PR. Both in vivo, using furin as the substrate
(Dittié et al., 1997) and in vitro, with both furin and the CIM6PR as substrate, we have found that phosphorylation of the
acidic cluster is the most important signal for recruitment of
AP-1 to ISGs. Phosphorylation of the acidic cluster may
regulate AP-1 binding to ISGs, and the removal of proteins
from ISGs in CCVs. For example, if during maturation the
dense core underwent a physical rearrangement, as
documented for insulin (Michael et al., 1987), it might be
advantageous only to remove proteins after this rearrangement
had occurred thus ensuring that all soluble non-granule
proteins were excluded from the dense-core.
AP-1 recruitment to ISGs was inhibited in competition
experiments using the phosphorylated cytoplasmic domains of
the CI-M6PR. These results extend our previous results with
furin (Dittié et al., 1997) and the results of Hoflack and
colleagues (Le Borgne et al., 1993) which showed that CKII
phosphorylation of the recombinant bovine CI-M6PR
cytoplasmic domain GST-fusion protein was required to
competitively inhibit AP-1 binding to TGN membranes. In
addition, our results demonstrate that the phosphorylated CIM6PR cytoplasmic tail can compete AP-1 recruitment to ISGs
more effectively than furin. This probably reflects the higher
affinity of AP-1 for the phosphorylated CI-M6PR cytoplasmic
domain rather than the amount of furin and M6PR in the ISGs.
Although the amount of M6PR present in the ISGs is difficult
to estimate we can detect both the CI- and CD-M6PR in the
ISG fraction by subcellular fractionation and the CD-M6PR by
immunogold labelling on cryosections. Interestingly, using
immunoisolation we found that at most only 50% of the ISGs
had significant enough amounts of CI-M6PR to be
immunoisolated, whereas most of the ISGs contained furin.
Our results raise the possibility that there is a heterogenous
populations of clathrin-coated vesicles forming from the ISGs
in PC12 cells. Clathrin-coated vesicles could form from ISGs
containing furin alone, or containing both furin and the M6PR.
However, in PC12 cells ISGs can undergo homotypic fusion as
part of their maturation to MSGs (Tooze et al., 1991; Urbé et
al., 1998) and this homotypic fusion is thought to be a
prerequisite for clathrin-coated vesicle formation (Tooze,
1991). If CCV formation is restricted to a late stage of granule
maturation, homotypic fusion may serve to ensure that all ISGs
have a uniform composition and that the CCVs emanating from
the ISGs will be a homogenous population. Irregardless of the
composition of the ISG derived CCVs it is most likely that these
CCVs will go to the endosome. The alternative possibilities that
M6PRs are found in a subset of PC12 cell ISGs 3965
the furin-only CCVs go to the TGN or plasma membrane would
require additional sorting steps, such as exclusion of M6PRs,
or the inclusion of targeting molecules (such as SNARES) into
the furin-only-CCVs. Such sorting steps, entailing exclusion
and/or inclusion of transmembrane molecules and cargo, are
usually mediated by coat-machinery. As both M6PR and furin
bind AP-1 these sorting steps cannot be mediated by AP-1.
Recently, proteins of a new class have been identified which
function as connectors between membrane proteins and
clathrin-coats, including PACS-1 (Wan et al., 1998; Molloy et
al., 1999). However, as PACS-1 binds to the CKII
phosphorylated acidic cluster which is present in furin as well
as the CI-M6PR, it could not provide the sorting machinery
required to generate two different CCVs from the ISG.
In other cell types, such as pancreatic β-cells, in which the
clathrin-coated ISGs do not appear to undergo homotypic
fusion, it will be interesting to determine if there is
heterogeneity between ISGs with respect to furin and the
M6PR. It may be that the concentration of the M6PR is higher
in these cells and consequently that all ISGs have M6PR, and
all CCVs derived from these ISGs deliver their cargo to the
endosomes. This would imply that soluble molecules such as
C-peptide go to endosomes and then to the cell surface. The
alternative possibility is that there is an additional secretory
pathway from the ISG for C-peptide. This hypothetical
pathway, which may correspond to the constitutive-like
pathway, would not transport furin or M6PR nor would it
involve an AP-1 based clathrin coat. Further insight into this
possibility could come from a closer examination of the
kinetics of C-peptide secretion to determine if it is direct to the
plasma membrane or via endosomes.
The authors thank J. Sandall (ICRF) and V. Oorschot (University
of Utrecht) for technical assistance. We thank Drs W. Brown, Cornell
University, USA and Annette Hille-Rehfeld, Georg-August
Universität, Göttingen, Germany for antibodies. The authors are
indebted to Dr Graham Warren, Giampietro Schiavo and John Tooze
for careful reading of the manuscript, helpful comments and
discussions. The authors also thank Dr Gary Thomas, Vollum
Institute, Oregon, USA for contributions and dicussion during this
work. This work was in part supported by a EU TMR network grant
(ERB-FMRXCT960023) to S. Tooze.
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