From the Institut für Virologie der
Philipps-Universität Marburg, Robert-Koch-Strasse 17, 35037 Marburg, Germany and the Institut de Biologie, EP CNRS 525, Institut Pasteur de Lille BP 447, 1, rue Professeur Calmette, 59021 Lille Cedex, France.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The eukaryotic subtilisin-like endoprotease furin
is found predominantly in the trans-Golgi network (TGN) and cycles
between this compartment, the cell surface, and the endosomes. There is experimental evidence for endocytosis from the plasma membrane and
transport from endosomes to the TGN, but direct exit from the TGN to
endosomes via clathrin-coated vesicles has only been discussed but not
directly shown so far. Here we present data showing that expression of
furin promotes the first step of clathrin-coat assembly at the TGN, the
recruitment of the Golgi-specific assembly protein AP-1 on Golgi
membranes. Further, we report that furin indeed is present in isolated
clathrin-coated vesicles. Packaging into clathrin-coated vesicles
requires signal components in the furin cytoplasmic domain which can be
recognized by AP-1 assembly proteins. We found that besides depending
on the phosphorylation state of a casein kinase II site, interaction of
the furin tail with AP-1 and its µ1subunit is mediated by a tyrosine
motif and to less extent by a leucine-isoleucine signal, whereas a
monophenylalanine motif is only involved in binding to the intact AP-1
complex. This study implies that high affinity interaction of AP-1 or
µ1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal.
The trans-Golgi network
(TGN)1 constitutes the
sorting station where soluble and membrane proteins, targeted to
different post-Golgi compartments, are packed into distinct carrier
vesicles. Three TGN exit routes are known: constitutive transport to
the cell surface, transport to secretory granules in cells with a
regulated secretory pathway, and transport to endosomes (for review,
see Ref. 1). The last pathway is followed primarily by lysosomal enzymes bound to the mannose 6-phosphate receptors (MPRs) by their mannose 6-phosphate residues (for review, see Ref. 2). Vesicles leaving
the TGN for subsequent transport to the endosomes are clathrin-coated.
Formation of these clathrin-coated vesicles (CCVs) involves binding of
AP-1 Golgi-specific assembly proteins to the TGN membrane. AP-1 binding
is regulated by the ADP-ribosylation factor ARF-1, a small GTP-binding
protein (3, 4), and requires the presence of transmembrane proteins,
among which MPRs are the major constituents (5-7). CCVs are transport
intermediates of vesicular traffic not only from the TGN to endosomes,
but also from the plasma membrane to endosomes. Golgi- and plasma
membrane-derived CCVs can be distinguished by their different sets of
assembly proteins; AP-1 complexes are restricted to coated buds and
vesicles of the TGN, whereas AP-2 complexes act in endocytosis at the
plasma membrane. Both adaptor complexes are heterotetrameric. The AP-1 complex is composed of two 100-kDa subunits, Furin is a membrane-associated, subtilisin-like eukaryotic endoprotease
that proteolytically cleaves a large number of proproteins C-terminally
at the consensus sequence RXK/RR. Among these substrates are
membrane-bound and secretory proteins such as prohormones, proproteins
of receptors, plasma proteins, growth factors, and also bacterial
toxins and many surface glycoproteins of enveloped viruses (27; for
review, see Refs. 28 and 29). Furin is concentrated in the TGN and
cycles between this compartment, the plasma membrane, and endosomes.
The responsible routing and TGN localization signals reside in the
cytoplasmic domain of furin (30-33). The sorting signals include the
tyrosine-based signal YKGL765, which mediates endocytosis,
and an acidic cluster CPSDSEEDEG783, which is required for
TGN localization (33-36). Deletion analysis and mutagenesis of the
furin tail revealed a leucine-isoleucine (LI760)
sorting signal and a critical phenylalanine (F790), which
separately mediate internalization of chimeric furin proteins.2 The two serine
residues of the acidic cluster are phosphorylated by casein kinase II
(CKII) in vivo and in vitro. Phosphorylation is
assumed to regulate trafficking of furin in endosomes (35, 36).
Recently, a cytosolic connector protein, PACS-1 (phosphofurin acidic
cluster-sorting proteins), was identified, which directs transport of
furin from the endosomes back to the TGN by binding to the
phosphorylated acidic cluster and connecting the endoprotease to the
clathrin sorting machinery (38). In the regulated secretory pathway of
neuroendocrine cells, retrieval of furin from clathrin-coated immature
secretory granules depends on the interaction of AP-1 with
CKII-phosphorylated furin tail (39). However, the routing of furin at
the exit of the TGN and especially the motifs required at this sorting
station are not well understood. In this study we show that furin is
able to promote AP-1 recruitment, the first step of clathrin coat
assembly at the TGN, and that furin is present in isolated CCVs. Using
in vitro binding and peptide competition assays, we have
examined in detail the interaction of the furin cytoplasmic tail
sorting signals with the µ1 subunit and the whole AP-1 complex.
Raising an Antiserum against the Cytoplasmic Domain of Bovine
Furin--
A cDNA fragment coding for the furin tail (amino acids
741-797) was amplified by polymerase chain reaction from pSG5:bfur (33) and ligated into the EcoRI-HindIII sites of
the Escherichia coli expression vector pMALTM-c2
(New England Biolabs, Germany). The plasmid was transfected into the
E. coli TB1 strain. The furin tail containing the
maltose-binding protein was purified by affinity chromatography
according to the protocol provided by Biolabs (Germany), dialyzed
against PBS, and used in 200-µg portions for the immunization of
rabbits by four injections at 4-week intervals. The serum was tested by
enzyme-linked immunosorbent assay, Western blotting,
immunoprecipitation, and immunofluorescence.
Recombinant DNA Methods--
The µ1 subunit of TGN adaptor
AP-1 (AP47) was cloned by polymerase chain reaction from human lung
GST Fusion Protein Production--
The cDNA coding for the
cytoplasmic domains of wild type furin and furin tail mutants was
ligated in-frame to the COOH terminus of GST using the pGEX-5X-1 vector
(Pharmacia, Germany). The fusion proteins were expressed in E. coli strain BL21, and protein purification was carried out
according to the manufacturer's instructions (Pharmacia, Germany).
Preparation of Bovine Brain Cytosol--
Bovine brain cytosol
was prepared as described (41). Briefly, bovine brains were homogenized
in buffer A (0.1 M MES, 0.5 mM
MgCl2, 1 mM EGTA, pH 7.0) containing 0.1 mM phenylmethylsulfonyl fluoride and 0.2 mM
dithiothreitol. Nuclei, membranes, and other sedimental components were
removed by centrifugation at 8,000 × g for 50 min at
4 °C followed by centrifugation at 130,000 × g for
1 h at 4 °C. The protein concentration of the cytosol
supernatant was determined by protein assay (Pierce Chemical Co.) using
BSA as a standard.
Isolation of CCVs--
CCVs were isolated from MPR-deficient
mouse fibroblasts (42) stably expressing furin by a standard procedure
(43, 7). After three washes with PBS containing 0.1 mM
CaCl2 and 1 mM MgCl2 (PBS-Ca/Mg),
cells were scraped and spun for 10 min at 2,000 × g.
The cells were resuspended in vesicle buffer (140 mM
sucrose, 75 mM potassium acetate, 10 mM MES, pH
6.7, 1 mM EGTA, 0.5 mM magnesium acetate)
containing 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and protease inhibitor mixture
(Boehringer Mannheim) and homogenized. From a postnuclear supernatant,
a postmitochondrial supernatant was prepared, which was fractionated by
two gradient centrifugations using a 10-ml continuous gradient of
10-90% (w/v) 2H2O (Sigma) in vesicle buffer
and then a 10-ml continuous gradient from 2% (w/v) Ficoll, 9% (w/v)
2H2O to 20% Ficoll, 90%
2H2O in vesicle buffer. After centrifugation of
the second gradient to equilibrium, 1-ml fractions were collected from
the bottom, and aliquots of each fraction were precipitated by
trichloroacetic acid (10% final concentration) for Western blotting analysis.
Generation of MPR-deficient Mouse Fibroblasts Stably Expressing
Furin---
The bovine furin subcloned into the pSG5 vector was
stably expressed in MPR-negative fibroblasts as described (18).
Briefly, a calcium phosphate precipitate was made containing 20 µg of
the linearized vector and 1 µg of pBhygr, a plasmid containing the hygromycin resistance gene. The cells were incubated with the precipitate for 24 h, split, and plated in the presence of 350 µg/ml hygromycin B (Boehringer Mannheim). After 12-14 days of selection, colonies were picked, expended, and tested for expression of
furin by immunofluorescence.
Cell Culture and Transfection--
HeLa cells or immortalized
MPR-deficient mouse fibroblasts (42) and MPR-deficient mouse
fibroblasts stably expressing bovine furin were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. For
transient expression, HeLa cells were transfected with pSG5:bfur
constructs by the calcium phosphate method (44). At 7 h
post-transfection protein expression was stimulated by the addition of
sodium butyrate (5 mM final concentration) to the culture medium.
Binding of AP-1 to Golgi Membranes--
The in vitro
binding of AP-1 was performed as described (5, 6, 17), except for using
a different permeabilization technique (45). MPR-negative mouse
fibroblasts stably expressing furin and MPR-negative mouse fibroblasts
as control cells were cocultured on coverslips for 24 h to 90%
confluence. Cells were washed twice with cold PBS-Ca/Mg and KOAc buffer
(25 mM Hepes/KOH, pH 7.0, 111.5 mM potassium
acetate, 2.5 mM MgCl2), permeabilized by
immersion in ice-cold KOAc buffer containing 40 µg/ml digitonin for 4 min, and then washed twice with cold KOAc buffer again. For AP-1
binding the permeabilized cells were incubated with 200 µl of bovine
brain cytosol (9 mg of protein/ml in KOAc buffer) at 37 °C for 10 min and replaced on ice for two washes with cold KOAc buffer. Then the
cells were subjected to immunofluorescence analysis.
Indirect Immunofluorescence and Image
Processing--
Furin-transfected HeLa cells were fixed with 4%
paraformaldehyde in PBS for 15 min and then quenched with 50 mM NH4Cl in PBS. For permeabilization, cells
were rinsed with 0.2% saponin (Sigma) that was dissolved in PBS and
then incubated with a furin-specific antiserum from rabbit directed
against the cytoplasmic tail (diluted 1:1,000) and
MPR-deficient mouse fibroblasts were fixed with 3% paraformaldehyde in
PBS for 15 min, quenched with 50 mM NH4Cl in
PBS, and then incubated with primary and secondary antibodies, diluted in PBS containing 1% BSA, as described for HeLa cells.
The intensity of the perinuclear In Vitro Binding Assays--
The µ1 chain was translated
in vitro in the presence of 35S-labeled
methionine (Amersham, Germany) using the pBluescript µ1 DNA as
template and a coupled in vitro transcription translation kit (Promega). In vitro translated samples were centrifuged
at 15,000 × g for 10 min to remove insoluble
materials. Glutathione-Sepharose 4B beads (Pharmacia Biotech, Sweden)
loaded with 30 µg of wild type or mutated GST-furin fusion protein
were incubated with or without CKII (25 units/µl) (Biolabs, Germany)
and 2 mM ATP in 30 µl of CKII buffer (20 mM
Tris-HCl, 50 mM KCl, 10 mM MgCl2, pH 7.5) for 1 h at 30 °C. Then precleared in vitro
translated µ1 was added followed by incubation for 2 h at
4 °C in 500 µl of binding buffer (0.05% Nonidet P-40, 50 mM Hepes, pH 7.3, 10% glycerol, 0.1% BSA, 200 mM NaCl). Sample buffer was added to washed beads, and the
eluted material was subjected to SDS-PAGE. The radiolabeled bands were
detected by fluorography. Quantitation was done by using a BioImager
(Raytest, Germany).
For binding of AP-1 to immobilized furin tails 30 µg of GST-furin
fusion protein were adsorbed to glutathione-Sepharose 4B and incubated
with or without CKII as described above. The immobilized fusion
proteins were suspended in 400 µl of bovine brain cytosol (6 mg of
protein/ ml in buffer A) containing 1 mM
phenylmethylsulfonyl fluoride and protease inhibitor mixture and
incubated for 1 h at 4 °C. Sepharose beads were washed three
times with cold buffer A (see above) and then resuspended in sample
buffer. The bound material was separated by SDS-PAGE and subjected to
immunoblot analysis with In Vitro Competition Assay--
Oligopeptides homologous to the
furin tail were synthesized by using an ABI 432A peptide synthesizer
(Applied Biosystems, Inc.). In vitro translated,
35S-labeled µ1 chain was preincubated in 100 µl of
binding buffer in the presence or absence of peptides for 15 min on
ice. Then immobilized GST-furin fusion protein was added and incubated
for 2 h at 4 °C. For AP-1 competition, 100 µl of bovine brain
cytosol (1 mg of protein/ml in buffer A) was preincubated with or
without peptides for 15 min on ice followed by incubation with
immobilized GST-furin fusion protein for 1 h at 4 °C. Detection
of bound AP-1 or µ1 was done as described above.
Western Blotting--
Electrophoretic transfer of proteins to
nitrocellulose membranes was done by standard procedure (46).
Incubations with primary antibodies diluted in PBS containing 0.1%
Tween 20 were performed for 1 h at room temperature. Primary
antibodies were detected using horseradish peroxidase-labeled
anti-mouse antibody from rabbit (Dako, Denmark), and the secondary
antibodies were detected using the Super Signal system (Pierce). When
blots were probed sequentially with different antibodies, the membranes
were stripped in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM Primary Antibodies--
The primary antibodies were: anti- Furin Recruits AP-1 onto Golgi Membranes--
Proteins like the
MPRs, which are transported from the TGN to the endosomes via CCVs,
have been shown to promote AP-1 recruitment onto Golgi membranes (5).
To prove sorting of furin along the TGN/endosomal pathway we
investigated first whether furin also promotes AP-1 translocation. For
this purpose, wild type furin (wt) and furin tail mutants lacking the
tyrosine-motif (dYKGL) or the cytoplasmic domain (d743-797) or one
that contains a nonphosphorylatable acidic signal (S776A/S778A) (Fig.
1A) were expressed in HeLa
cells. To discriminate adaptin recruited by furin from adaptin
recruited by other Golgi resident proteins, furin expression was
stimulated by the addition of sodium butyrate. Then the cells were
double labeled with a polyclonal anti-furin antibody to discriminate transfected from nontransfected cells, and with a monoclonal antibody directed against
In a second assay we investigated recruitment of AP-1 by furin at a
lower and thus more physiological level of furin expression. We took
advantage of the MPR-negative fibroblasts generated and described by
Ludwig et al. (42). These cells exhibit a low capacity for
recruiting AP-1, which can be restored by reexpressing physiological levels of the MPRs (6). MPR-deficient mouse fibroblasts stably expressing furin and MPR-deficient mouse fibroblasts as control were
cocultured, permeabilized with digitonin, and incubated with bovine
brain cytosol. Recruitment of bovine AP-1 was examined by indirect
immunofluorescence using a polyclonal anti-furin antibody and
monoclonal antibody 100/3 against
These results imply that furin, which is predominantly localized in the
TGN, is able to recruit AP-1. Recruitment seems to be dependent on the
presence of YKGL765 and the phosphorylated acidic motif
CPSDSEEDEG783.
Furin Localizes to CCVs--
The observed recruitment of AP-1 on
membranes of the Golgi region by furin is consistent with the sorting
of furin into CCVs at the TGN. However AP-1 binding does not
necessarily prove that furin is also sorted into CCVs in
vivo. Therefore, using a standard fractionation protocol (7, 43),
CCVs were prepared from the MPR-deficient mouse fibroblasts (42) stably
expressing furin. The last density gradient of the purification
procedure was analyzed by Western blotting. Fig.
3 shows the typical distribution of different marker proteins throughout this gradient. As described (7),
the dense fractions contained clathrin heavy chain, The Cytoplasmic Tail Signals YKGL765,
LI760, F790, and Clustered Acidic Amino Acids
Are Required for Furin Interaction with AP-1 Golgi-specific Assembly
Proteins in Vitro--
Interaction of the cytoplasmic domain of
different proteins with adaptor complexes and their medium (µ) chains
has been shown. Most of these interactions are mediated by
tyrosine-based motifs and also leucine-based motifs. As shown by others
(39) the TGN adaptor AP-1 binds to the cytoplasmic tail of furin
in vitro dependent on the phosphorylation state of a CKII
site within an acidic cluster. Our recruitment experiments described
above show that furin expression promotes AP-1 binding to Golgi
membranes. They indicate further that the phosphorylated motif
CPSDSEEDEG783 and in addition the tyrosine-based motif
YKGL765 are determinants for AP-1 binding in
vivo. It has been shown previously that, besides the tyrosine and
the CKII acidic motif (33-36), two other determinants, a
leucine-isoleucine sorting signal LI760 and a
monophenylalanine motif F790 (see Footnote 2) are critical
for correct intracellular sorting of furin. Therefore we tested the
role of these four signals for interaction with AP-1 or its µ1
component in vitro. A set of furin tail mutants was produced
substituted in the tyrosine, the LI, or the phenylalanine motif, and
also in the cluster of acidic amino acids. These mutants have been
analyzed in the following experiments.
AP-1 Binding to the Furin Tail Requires a Tyrosine-based Motif, a
LI Sorting Signal, and, in Addition, a Monophenylalanine Motif--
We
tested whether the AP-1 adaptor complex can bind to the cytoplasmic
tail of furin and furin tail mutants (Fig.
4A). GST-furin fusion proteins
(GST-F) containing the cytoplasmic tail of wild type (wt) furin or
mutated furin were used for this purpose. GST-F were immobilized on
glutathione-Sepharose and incubated either with or without CKII to
obtain phosphorylated or nonphosphorylated versions of the furin tail.
Then bovine brain cytosol was added as the source of adaptor complexes,
and the amount of AP-1 bound was measured by immunoblotting with the
bovine-specific anti µ1 Binding to the Furin Tail Requires a Tyrosine-based Motif and
a LI Sorting Motif--
Next we tested binding of the µ1 chain of
AP-1 to the furin tail. Again we probed GST-F containing the
cytoplasmic tail of wild type and mutated furin (Fig. 4A) in
the phosphorylated or nonphosphorylated version. In vitro
translated, [35S]methionine-labeled µ1 chain was added,
and the amount of bound µ1 was measured by autoradiography. As shown
in Fig. 4C the medium chain µ1 bound to GST-Fwt but not to
GST alone. Used as a negative control, in vitro translated
influenza virus NS1 did not interact with GST-Fwt (data not shown).
Further, furin tail lacking either the tyrosine motif or the LI motif,
mutants AKGA and LI/AN, bound 3-fold or 2-fold less efficiently µ1
than wild type. When the phenylalanine 790 had been mutated to
asparagine (mutant F/N) interaction with µ1 was as efficient as for
the furin wild type. This is in contrast to the AP-1 binding behavior
with the F/N mutant (see Fig. 4B). The double mutants AKGA
F/N and LI/AN F/N interacted with µ1 like the single mutants AKGA and
LI/AN, also indicating that substitution of phenylalanine 790 does not
influence the binding of µ1 chain. After substitution of both the
tyrosine-based motif and the LI motif (mutant LI/AN Y/A) and as
expected, when all three motifs were destroyed (mutant LI/AN Y/A F/N),
practically no interaction was measured. As already found for AP-1
binding, we observed that phosphorylation of wild type and mutated
furin resulted in an about 2-fold increase in µ1 binding. To ensure that increased µ1 binding to the furin tail is really caused by phosphorylation, we dephosphorylated CKII-treated GST-Fwt with alkaline
phosphatase. After phosphatase treatment the µ1 binding was reduced
again, showing that stronger interaction attributes to CKII
phosphorylation (data not shown). From these experiments it was
apparent that the tyrosine-based motif and the LI sorting motif
determine interaction of the furin tail with the medium chain µ1.
Phosphorylation of Ser776 and Ser778 by CKII
enhances this interaction.
The Number of Negative Charges in the Acidic Cluster Is Crucial for
the Affinity of AP-1 Binding--
The effect of CKII phosphorylation
on AP-1 binding raises the question of whether phosphates at a distinct
site or an overall negative charge is critical for the enhancement of
AP-1 binding affinity. Furin is phoshorylated by CKII both in
vivo and in vitro on Ser776 and
Ser778, and exchange of both serine residues to aspartic
acid mimics the diphosphorylated state of furin (35, 39). As expected, such a GST-furin mutant (mutant S776D/S778D, Fig.
5A) bound Furin Tail Peptides Specifically Compete for Furin AP-1 and µ1
Interaction--
Although the specificity of AP-1 and µ1 binding to
the furin tail were apparent from the effect of the single amino acid
substitutions, we confirmed the specificity and the requirement of the
different motifs by peptide competition assays. Therefore we tested the ability of furin tail homologous peptides (Fig.
6A) to inhibit the interaction
of AP-1 or µ1 with GST-Fwt (see Fig. 4A) in our in
vitro binding assays. After a preincubation of bovine brain cytosol or in vitro translated µ1 with increasing amounts
of competing peptides, we determined the amount of AP-1 or µ1 that
was still able to bind to GST-Fwt. The competition curves in Fig. 6,
B and C, show that the phosphorylated 41-amino
acid tail peptide Arg757-Leu797 inhibits the
binding of Furin is found predominantly in the TGN at steady state and
appears to cycle between this compartment, the plasma membrane, and the
endosomes. Experimental evidence for recycling has been obtained from
immunofluorescence-based assays indicating that furin is endocytosed
from the plasma membrane and travels from endosomes to the TGN
(30-33). Furin can enter the regulated pathway of secretory cells but
is subsequently removed from immature secretory granules by AP-1
containing CCVs (39). However, the direct exit from the TGN to
endosomes in nonsecretory cells has only been discussed (33, 34, 38),
but not directly shown so far. This pathway would probably involve
AP-1-containing CCVs budding from the TGN. Furthermore, as a specific
rather than a default pathway of furin transport, it may require signal
components in the furin cytoplasmic domain which mediate the packaging
into these CCVs. This prompted us, first, to see whether there was
evidence for AP-1/clathrin-dependent sorting of furin at
the TGN and to analyze then in detail the binding sites in the
cytoplasmic domain required for the interaction with AP-1 assembly proteins.
In this study we report that furin expression promotes the
translocation of AP-1 assembly proteins onto Golgi membranes of HeLa
cells and MPR-deficient mouse fibroblasts. AP-1 recruitment has been
shown for the MPRs (5-7), which follow the TGN/endosomal pathway (47)
and determine the amount of membrane-bound AP-1 and the number of
TGN-derived CCVs (7). Minor components in transit through the TGN, the
major histocompatibility complex class II and the varicella-zoster
virus glycoprotein I, have also been demonstrated to trigger AP-1
recruitment on Golgi membranes, suggesting that they can potentially be
packed in the same TGN-derived CCVs as the MPRs (17, 48). Of course,
AP-1 recruitment does not necessarily prove the sorting into CCVs
in vivo. However, we show here that furin is present in CCVs
isolated from furin-expressing MPR-deficient mouse fibroblasts. The
applied method for CCV isolation does not permit distinguishing CCVs
derived from the plasma membrane from those derived from the TGN. But
together with the finding that furin also promotes AP-1 recruitment in
the MPR-deficient fibroblasts, it is unlikely that furin is only
a component of plasma membrane-derived CCVs. Thus, our results now
strongly suggest that furin is packaged in AP-1-containing CCVs derived
from the TGN. This notion is also supported by previous morphological
studies suggesting that furin localizes to clathrin-coated regions of the TGN (33, 34).
The AP-1 recruitment experiments suggested that the tyrosine motif and
the phosphorylated acidic signal play a role in AP-1/furin binding
in vivo. For exact analysis of the requirement of furin cytoplasmic tail sorting signals YKGL765,
LI760, F790, and phosphorylated acidic cluster
CPSDSEEDEG783 for interaction with AP-1 and its µ1
subunit we made use of an in vitro binding assay. Tyrosine
or dileucine motifs in the cytoplasmic tails of membrane proteins have
been reported to interact with AP-1 and AP-2 in similar experiments
(49, 14, 13). The µ chains of adaptor complexes were also shown to
recognize a variety of tyrosine-based motifs (15, 22-24) or dileucine
motifs (25, 26) in the yeast two-hybrid system and a number of in
vitro assays. On the other hand, dileucine motifs have been
reported to interact with Our results show, first, that furin/AP-1 and µ1 association strongly
depends on the tyrosine motif YKGL765 and to less extent on
the leucine-isoleucine signal LI760. The LI motif may
therefore be different from classical dileucine motifs that have been
shown to interact with Second, we found that the monophenylalanine motif F790 is
only involved in binding to the intact AP-1 complex. Sorting signals that contain phenylalanine are rarely described, and their
participation in adaptor binding has not been reported previously.
Substitution of phenylalanine 790 does not affect the interaction
between µ1 and the furin tail. The reason for this discrepancy is
unclear at present. Two hypotheses could be envisaged. First, it is
possible that µ1 is involved in binding F790, but only
when present in the context of the complete AP-1 complex. An
alternative explanation could be that F790 binds to a
different subunit on the AP-1 complex. It has been suggested that the
Further, our data confirm that phosphorylation of the two CKII sites
within the acidic cluster is important for high affinity AP-1 binding
to the furin tail (39), but we clearly demonstrate that phosphorylation
enhances binding only in the presence of an intact tyrosine and
phenylalanine motif. We show that phosphorylation also correlates with
an increase in µ1 binding. Neither the phosphorylated nor the
nonphosphorylated furin tail is capable of binding to µ1 after the
loss of YKGL765 and LI760. These data suggest
that phosphorylation of the acidic cluster modulates the interaction
between furin and AP-1/µ1 rather than providing a specific binding
site. The two phosphate groups may function directly in interaction
with AP-1 or indirectly by regulating the accessibility of sorting
motifs, for example YKGL765 or LI760.
Alternatively, it is possible that phosphorylation of the CKII sites in
the cluster of acidic amino acids increases the affinity for AP-1 by
providing additional negative charges. It has been demonstrated that
exchange of both serine residues, Ser776 and
Ser778, for aspartic acid mimics the diphosphorylated state
of furin (35, 39). We show now that for efficient AP-1 binding it is irrelevant whether negative charges are introduced by substitution of
Ser776 and Ser778 to aspartic acid or by adding
acidic amino acid residues to other positions within the acidic
cluster. Our results argue that high affinity AP-1 binding is dependent
on the number of negative charges within the acidic sequence but not on
the overall charge of the furin tail. Mauxion et al. (18)
reported for the cation-dependent MPR that the entire CKII
phosphorylation site in the cytoplasmic domain is a dominant TGN
sorting determinant for efficient packaging of the receptor in
Golgi-derived vesicles. Similar to our observations these authors found
that replacement of three negatively charged amino acids surrounding
the serine at position 57 by alanine residues impaired AP-1 binding to
membranes. On the other hand, replacement of the positively charged
arginine at position +3 of the serine 57 in the cation-dependent
MPR tail by an aspartic acid resulted in an increased affinity of
AP-1 for membranes.
Recently, Wan and co-workers (38) identified a new cytosolic connector
protein, PACS-1, which binds specifically to the phosphorylated acidic
cluster and connects furin to the AP-1/clathrin sorting machinery for
subsequent transport from the endosomes back to the TGN. Our in
vitro data suggest that there is also a direct interaction of the
furin cytoplasmic domain with AP-1 and µ1. The interaction requires
combination of the furin tail sorting signals YKGL765,
LI760, F790, and phosphorylated acidic cluster
rather than one distinct signal. However, it is worth mentioning that
the bovine brain cytosol used in our study as source of AP-1 complexes
might contain PACS-1, which thus mediates the furin-AP-1 interaction by
binding to the phosphorylated acidic cluster. The in vitro
translated samples could also contain some PACS-1 derived from the
reticulocyte lysate of the translation kit. But, if so, we would expect
that destruction of YKGL765, LI760, or
F790 would not affect AP-1 or µ1 binding. Therefore, two
different mechanisms of furin/AP-1 association may exist: a direct
interaction mediated by combination of the different furin tail sorting
signals at the TGN and a PACS-1-mediated interaction dependent on
phosphorylation of the two CKII sites at endosomes.
In summary, our studies have shown that besides the phosphorylation
state of a CKII site, interaction of the furin tail with AP-1 or µ1
depends on a tyrosine motif and to less extent on a leucine-isoleucine
signal, whereas a phenylalanine motif (F790) is only
involved in binding to the intact AP-1 complex. Whether in
vivo all signals are utilized in TGN sorting of furin and whether additional cytosolic proteins are involved in furin-AP-1 interaction at
the exit of the TGN remain to be determined. However, the results imply
that high affinity interaction of AP-1 with the cytoplasmic tail of
furin needs a complex interplay of signal components rather than one
distinct signal.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
and
1 adaptin, a
medium subunit µ1 (47 kDa), and a small subunit
1 (19 kDa). The
AP-2 complex is of similar size and composition with two large subunits,
and
2 adaptin (100 kDa), in association with a µ2 (50 kDa) and a
2 subunit (17 kDa) (for review, see Ref. 8). A major
function of both adaptor complexes is to promote clathrin cage assembly
onto the respective membrane via their
subunits (9). A second
important function of AP-1 and AP-2 is the binding to cytoplasmic
domains of membrane proteins (for review, see Ref. 10) by interaction
with their tyrosine-based motifs (11-15) or dileucine-based sorting
signals (13, 16-19). Both motifs are known to be important for
endocytosis at the plasma membrane as well as targeting to various
endosomal compartments, to lysosomes, and to the basolateral plasma
membrane in polarized cells (for review see Refs. 20 and 21). With the
yeast two-hybrid system and a number of in vitro assays it
was shown that different types of tyrosine-based sorting signals (15,
22-24) and also dileucine motifs (25, 26) can be recognized by the µ chains of AP-1 and AP-2.
EXPERIMENTAL PROCEDURES
gt10 cDNA library (CLONTECH, USA). The
primers were derived from the mouse sequence (M62419) (40): forward
primer, 5'-CCTTCCAAAGCCGCCATGTCCGCCAGCGCCGTC-3'; reverse primer,
5'-CAGAGGCCTCTCACTGGGTCCGGAGCTGATAATC-3'. The polymerase chain reaction
product was ligated in the TA vector (Invitrogen) and sequenced by the
dideoxy chain method using the ABI PRISMTM Dye Terminator
Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). After excision by
EcoRI, the cDNA of the µ1chain was inserted into the
multiple cloning site of pBluescript vector (Stratagene). For
generation of the different furin tail mutants the pSG5:bfur construct,
described by Schäfer et al. (33), was used.
Substitutions in the cytoplasmic tail were introduced by a polymerase
chain reaction-based approach using pSG5:bfur as a cDNA template
and synthetic oligonucleotides that contained the desired mutations within their sequence. Generation of the deletion mutants was done as
described by Schäfer et al. (33). The sequences of all
of the tail mutants were verified by dideoxy sequencing.
adaptin-specific
monoclonal antibody 100/3 (diluted 1:100) (Sigma) diluted in PBS
containing 0.2% saponin and 1% BSA. The primary antibodies were
detected by incubation with rhodamine-conjugated anti-mouse
antibody from goat and fluorescein isothiocyanate-conjugated anti-rabbit antibody from goat (Dianova, Germany), both diluted 1:200
in PBS containing 1% BSA. Finally, the cells were mounted in Mowiol
(Hoechst, Germany) and 10% 1,4-diazabicyclo[2.2.2]octane.
adaptin fluorescence signal was
quantitated on the single cell level. Digitized images were recorded
using a laser scanning microscope (Carl Zeiss, Oberkochen, Germany)
working with the blue line (408 nm wavelength) of an argon laser. For
each sample, images of about 100 furin-expressing cells and as control
a similar number of furin negative cells were recorded and saved as
TIFF files. The intensity of perinuclear staining for
adaptin was
quantitated using the PC BAS software (Raytest, Germany).
adaptin-specific monoclonal antibodies.
-mercaptoethanol for 30 min at 50 °C after each
immunodetection. Quantitation of the signals was done with a BioImager.
adaptin monoclonal antibody against mouse
adaptin, anti-
adaptin
monoclonal antibody generated from mouse
A adaptin,
anti-clathrin heavy chain monoclonal antibody generated from rat
clathrin H. C. (Transduction Laboratories), anti-
adaptin
monoclonal antibody 100/3 against AP-1 adaptor from bovine brain,
anti-
-COP monoclonal antibody (Sigma), and mon139 monoclonal
antibody against the cytoplasmic domain of furin (a kind gift from Dr.
J. Creemers, Leuven).
RESULTS
adaptin to identify the AP-1 complex. Quantitation of AP-1 recruitment by measuring the fluorescence associated with the
Golgi region (Fig. 1C) and qualitative analysis of AP-1
recruitment (Fig. 1B, a-h) have shown that
overexpression of wild type furin correlates with an increase of
adaptin staining in the perinuclear region. Cells expressing the furin
mutants dYKGL and S776A/S778A show only a slight increase in a
perinuclear
adaptin signal, whereas the tail-less furin mutant
(d743-797), which was also found partly in the perinuclear region
because of its overexpression, remains without any significant effect
on adaptin binding. As a control, highly expressed influenza virus
hemagglutinin, a protein known to follow the constitutive secretory
pathway toward the plasma membrane, showed no specific increase in the
perinuclear
adaptin signal (data not shown).
View larger version (49K):
[in a new window]
Fig. 1.
Recruitment of AP-1 in HeLa cells expressing
wild type furin and furin tail mutants. Panel A,
schematic representation of wild type furin and furin tail mutants. The
C-terminal amino acid sequences of wild type furin and mutated furin
are shown in one-letter code. The open bar symbolizes the
C-terminal part of the putative membrane anchor. The four sorting
motifs are underlined or marked by dashes.
Substituted amino acids are marked by arrowheads.
Panel B, HeLa cells were transfected with wild type furin
and furin tail mutants (a-h). Protein expression was
stimulated by the addition of sodium butyrate to the culture medium.
Cells were permeabilized, fixed, and double labeled with an antiserum
raised against the cytoplasmic tail of furin (a,
c, e, g) and with adaptin-specific monoclonal
antibody 100/3 (b, d, f, and
h) and immunostained. Panel C, the intensity of
the perinuclear
adaptin fluorescence signal was quantitated for
about 100 transfected and a similar number of nontransfected cells. The
ratio of these two fluorescence intensities was taken as the measure of
increase in adaptin signal. Three independent experiments were
performed. Error bars represent the S.D.
adaptin. As shown in Fig. 2, cells expressing furin exhibit a
markedly enhanced perinuclear
adaptin signal compared with
nontransfected fibroblasts (Fig. 2, a and b).
When bovine brain cytosol was omitted from the incubation, no AP-1 was
detected because the antibody does not recognize mouse adaptin (Fig. 2,
c and d). Quantitation of the
adaptin
fluorescence signal associated with the Golgi region revealed a
40-55% increase in adaptin binding for furin expressing MPR-deficient
fibroblasts compared with furin-negative control cells.
View larger version (129K):
[in a new window]
Fig. 2.
AP-1 binding in MPR-negative fibroblasts
stably expressing furin. MPR-negative fibroblasts stably
expressing wild type furin (arrowheads) and MPR-negative
fibroblasts (*) were cocultured on coverslips, permeabilized
with digitonin, and incubated with bovine brain cytosol (a
and b) or with corresponding buffer lacking cytosol as
control (c and d). Cells were fixed and processed
for double immunofluorescence using a polyclonal antibody against the
cytoplasmic tail of furin (a and c) and
monoclonal antibody 100/3 against bovine adaptin (b and
d).
adaptin, and
adaptin, whereas
-COP, a subunit of the coatomer chosen as a
marker for vesicles of the early secretory pathway, was detected in the
lighter fractions of the gradient. We detected furin in the same dense
fractions also containing the marker proteins for CCVs. Compared with
CCVs isolated from bovine brain by electron microscopy, the dense
fractions contained the same spherical structures of 50-100 nm in
diameter (not shown). The results demonstrate that furin is sorted into
CCVs in vivo.
View larger version (32K):
[in a new window]
Fig. 3.
Detection of furin in clathrin-coated
vesicles. CCVs were purified by a standard method (7, 43) from
MPR-negative fibroblasts stably expressing furin. Each fraction from
the second linear density gradient was subjected to SDS-PAGE and
analyzed by Western blotting for its content in clathrin heavy chain
(clathrin H.C.), adaptin,
adaptin, furin, and
-COP.
adaptin antibody 100/3. Confirming the results
of Dittié and co-workers (39), nonphosphorylated GST-Fwt bound
low amounts of
adaptin, but after phosphorylation by CKII, binding
increased (Fig. 4B). Several new findings obtained from our
furin tail mutants now demonstrate that the situation is more complex
(Fig. 4B). Mutants AKGA and LI/AN, substituted in the
tyrosine-based motif YKGL765 or the leucine-isoleucine
signal LI760, respectively, showed a significant decrease
in
adaptin binding compared with wild type furin. Surprisingly,
mutation of the phenylalanine 790 (mutant F/N) resulted in a total loss
of AP-1 interaction. Consistently, furin tail mutants substituted in
more than one motif (mutants AKGA F/N, LI/AN F/N, LI/AN Y/A, LI/AN Y/A
F/N) were also incapable of interacting with AP-1 adaptor complexes. Mutant LI/AN is the only mutant in which the binding affinity for AP-1
is still increased by phosphorylation, but only to about 60% of wild
type. The results of this experiment demonstrate that a tyrosine-based
motif, a LI sorting motif, and a single phenylalanine residue determine
interaction of AP-1 with the cytoplasmic tail of furin. The
phosphorylated acidic motif is important for high affinity binding but
is functional only in the presence of an intact tyrosine motif and
phenylalanine 790.
View larger version (33K):
[in a new window]
Fig. 4.
Binding of medium chain
µ1 and AP-1 to immobilized furin tail mutants.
Panel A, schematic representation of the furin portion of
GST-furin fusion proteins (GST-Fwt, GST-F AKGA, GST-F LI/AN,
GST-F F/N, GST-F AKGA F/N, GST-F LI/AN F/N, GST-F LI/AN Y/A, GST-F
LI/AN Y/A F/N). The amino acid sequences of the cytoplasmic tail of
wild type furin and furin tail mutants are shown in one-letter code.
The open bar symbolizes the GST portion of the GST-furin
fusion proteins. The four sorting motifs are underlined or
marked by dashes. Substituted amino acids are marked by
arrowheads. Panel B, GST, GST-Fwt, and GST-furin
tail mutants were immobilized on glutathione-Sepharose 4B beads,
phosphorylated (+) or not ( ) with CKII, and then incubated with
bovine brain cytosol. After several washes of the beads the bound
material was separated by SDS-PAGE, and the amount of AP-1 bound was
detected by immunoblotting with
adaptin-specific monoclonal
antibody 100/3 and quantitated by bioimager analysis. Panel
C, GST and GST-F loaded glutathione beads were incubated with
in vitro translated and [35S]methionine
labeled µ1 chain, and the bound material was separated by SDS-PAGE
and quantitated by bioimager analysis. The amount of bound µ1 chain
and AP-1, respectively, is expressed as a percentage of binding to CKII
phosphorylated wild type furin tail (GST-Fwt-P). Images of
representative experiments are shown in panels B and
C. At least three independent experiments were performed.
Error bars represent the S.E.
adaptin as
efficiently as CKII-phosphorylated GST-Fwt (Fig. 5B). Therefore we tested whether introduction of negative charges in the
form of aspartic acid also increases the affinity for AP-1 binding when
they are placed at different positions in the furin tail. Mutant
D798/799, a GST-F in which two aspartate residues were added to the
carboxyl terminus of the furin tail, interacted less efficiently with
adaptin than the mutant S776D/S778D but has a moderately higher
AP-1 interaction than nonphosphorylated wild type. Interestingly, the
R784D/G785D mutant, in which Arg784 and Gly785
were mutated to aspartic acid, bound to AP-1 as much as mutant S776D/S778D (see Fig. 5, A and B). These
observations imply that for efficient AP-1 binding it makes no
difference whether the negative charges are introduced by
phosphorylation of Ser776 and Ser778 within the
acidic sequence or by additional acidic amino acids in this region.
However, negative charges added far from the acidic sequence have much
less influence on
adaptin binding. Therefore high affinity AP-1
binding seems to be dependent on the number of negative charges in or
near the acidic cluster.
View larger version (27K):
[in a new window]
Fig. 5.
Binding of AP-1 to immobilized furin tail
mutants imitating the phosphorylated state of furin. Panel
A, schematic representation of the furin portion of GST-furin
fusion proteins (GST-Fwt, GST-F S776/778D, GST-F D798/799,
GST-F R784/G785D). Substituted and inserted amino acids are marked by
arrowheads. For details, see Fig. 4. Panel B,
glutathione-Sepharose 4B beads loaded with GST, GST-Fwt (phosphorylated
or not (+/ ) with CKII), and different GST-furin tail mutants were
incubated with bovine brain cytosol. The bound material was subjected
to SDS-PAGE. AP-1 detection and quantitation were done as described in
the legend of Fig. 4.
adaptin and µ1 to GST-Fwt most efficiently. Inhibition
was dose-dependent and occurred at micromolar
concentrations of peptide. As expected, inhibition of µ1 binding to
GST-F S776D/S778D, imitating the diphosphorylated state of furin, by
the peptide Arg757-Leu797 was much weaker (data
not shown). To narrow down the sequence required for the competitive
inhibition of adaptor binding, a short furin tail peptide
Asp756-Pro767 containing only the tyrosine
motif and the LI sorting motif was tested. The peptide
Asp756-Pro767 inhibited binding of AP-1 and its
µ1 component, but to less extent than the 41-amino acid tail peptide.
On the other hand, no competition was observed with two short peptides
Asp756-Pro767 AKGA and
Asp756-Pro767 LI/AN, substituted either in the
tyrosine motif or the LI sorting motif. This underlines the specificity
and cooperation of both motifs for
adaptin and µ1 binding. Taken
together, the peptide competition assays have shown that the tyrosine
motif and the LI sorting motif are critical determinants for
competition of furin interaction with AP-1 and µ1.
View larger version (26K):
[in a new window]
Fig. 6.
Competition of furin tail homologous peptides
for interaction of the furin tail with AP-1 and
µ1. Panel A, amino acid sequence of
the furin tail homologous peptides. Panel B, GST-Fwt was
immobilized on glutathione-Sepharose and incubated with bovine brain
cytosol in the absence or presence of various concentrations of the
peptides Arg757-Leu797 (R757-L757)
( ), Asp756-Pro767 (D756-P767)
(
), Asp756-Pro767 (D756-P767
LI/AN) (
), or Asp756-Pro767
(D756-P767 AKGA) (
). The bound material was separated by
SDS-PAGE and detected as described in the legend of Fig. 4. Panel
C, glutathione-Sepharose beads loaded with GST-Fwt were incubated
with 35S-labeled µ1 chain in the presence or absence of
the furin tail peptides (see panel B). Bound µ1 chain was
detected as described on Fig. 4. Data are represented as the percentage
of
adaptin or µ1 bound in the absence of the competing peptides.
Error bars represent the S.D.
DISCUSSION
1 adaptin (37); but, until now little was known about adaptor binding to proteins like furin, which contain multiple sorting components in their cytoplasmic tail. It was therefore
interesting to investigate if each of the motifs would be able to
contribute to AP-1 binding, and, if so, whether they would function
independently or in combination. Dittié and co-workers (39)
showed in vitro that AP-1 binding to the cytoplasmic domain of furin depends on CKII phosphorylation of the acidic cluster. We
demonstrate here that the situation for furin tail interaction with
AP-1 and its µ1 subunit is more complex.
1 adaptin (52). In fact, the
leucine-isoleucine motif LI760 is very close to the
tyrosine motif YKGL765 and could well be part of it.
Further evidence for the requirement of YKGL765 and
LI760 for AP-1 and µ1 binding is given by peptide
competition. As also found by others (14, 25, 12, 23), inhibition
required a relatively large molar excess of soluble peptides. This is
probably because only a minor fraction of the free peptides has the
proper conformation, or this may reflect differences in the affinity of
µ1 and AP-1 for soluble versus immobilized peptides. It is noteworthy that association of the furin-derived tyrosine motif ISYKGL
and µ1 was not observed in the yeast two-hybrid system (23), thus
demonstrating the importance of analyzing the binding components in the
context of the whole cytoplasmic domain.
subunits may be involved in the recognition of receptor tails,
based on studies in which the asialoglycoprotein receptor (50) and the
epidermal growth factor receptor (51) were found to bind to
adaptin. In addition, Rapoport and co-workers (37) reported recently
binding of dileucine motifs to
1 adaptin.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Camps for providing the MPR-negative fibroblasts stably expressing furin and R. Le Borgne for discussion and for critical reading of the manuscript. We thank A. Di Carlo for help with the furin tail mutants and Dr. P. Elsässer (Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Germany) for help with the confocal laser scanning microscope. We also gratefully acknowledge Dr. J. Creemers (University Leuven, Belgium) for the generous gift of the mon139 monoclonal antibody. Peptides were synthesized and kindly provided by Dr. M. Krause (Institut für Molekularbiologie und Tumorforschung, Universität Marburg, Germany).
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 286).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-6421-28-5145; Fax: 49-6421-28-8962; E-mail: garten{at}mailer.uni-marburg.de.
2 A. Stroh, W. Schäfer, S. Berghöfer, M. Eickmann, M. Teuchert, I. Bürger, H.-D. Klenk, and W. Garten (1999) Eur. J. Cell Biol., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TGN, trans-Golgi network; MPR(s), mannose-6-phosphate receptor(s); CCV(s), clathrin-coated vesicle(s); AP(s), assembly proteins(s); CKII, casein kinase II; PACS-1, phosphofurin acidic cluster-sorting protein-1; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; wt, wild type; GST-F, GST-furin fusion proteins.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|