From the Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Here we report the identification and
characterization of AP-4, a novel protein complex related to the
heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein
sorting in the endocytic and late secretory pathways. The key to the
identification of this complex was the cloning and sequencing of two
widely expressed, mammalian cDNAs encoding new homologs of the
adaptor Sorting of integral membrane proteins at various stages of the
endocytic and secretory pathways is mediated by interactions of signals
contained within the cytosolic domains of these proteins with
organellar coats associated with the cytosolic face of membranes (reviewed in Refs. 1-3). Two types of signals referred to as "tyrosine-based" and "dileucine-based" have been implicated in various sorting processes in mammalian cells, including internalization from the plasma membrane, lysosomal targeting, delivery to the basolateral plasma membrane of polarized epithelial cells, transport from early to late endosomes, and localization to specialized organelles such as melanosomes and antigen-processing compartments. Both tyrosine- and dileucine-based signals have been shown to interact
with heterotetrameric adaptor protein
(AP)1 complexes, which
associate with the protein clathrin and accessory molecules to generate
coated transport vesicles (1-3).
To date, three adaptor complexes, known as AP-1, AP-2, and AP-3, have
been identified (reviewed in Ref. 4). These complexes consist of two
large chains ( AP-1 plays a role in protein sorting from the TGN and endosomes to
compartments of the endosomal/lysosomal system, while AP-2 is involved
in clathrin-mediated endocytosis (1-4). AP-3 is associated with
endosomes (7, 11) and/or the TGN (15) and recruits integral membrane
proteins for transport to lysosomes and lysosome-related organelles
(16, 17). The existence of more sorting events mediated by tyrosine-
and dileucine-based signals than known AP complexes, however, suggests
the existence of additional adaptors remaining to be identified (3).
In this study, we have found through searches in EST data bases several
cDNAs that encode new mammalian homologs of AP subunits. Antibodies
raised to these homologs and to the recently described AP µ-related
protein, µ-ARP2 (18), allowed us to identify a novel heterotetrameric
adaptor-like complex, which we named AP-4. Like other AP complexes,
AP-4 is composed of two large chains ( Sequencing of Northern Blotting--
Northern blot analysis was performed on
multiple tissue blots (CLONTECH) as described
previously (7). Both the GST Fusion Proteins--
A GST- Antibodies--
Polyclonal antibodies to Biochemical Procedures--
All of the biochemical procedures
used in this study have been described in a previous report (7).
Further details on the immunoprecipitation-recapture method are given
elsewhere (23). The sedimentation coefficient of AP-4 was estimated
from sedimentation velocity experiments using the following protein
markers (s20, w values given in
parentheses): horse spleen ferritin (16.5 S), bovine catalase (11.3 S),
BSA (4.6 S), and chicken ovalbumin (3.6 S) (24). The gel filtration
markers used to determine Stokes radii were those described previously
(7). The molecular mass of the AP-4 complex was calculated on the basis
of its Stokes radius and sedimentation coefficient, assuming a partial
specific volume of 0.72-0.75 cm3/g (24).
Immunofluorescence Microscopy--
HeLa cell monolayers were
grown on glass coverslips and in some cases treated with brefeldin A or
transiently transfected with HA epitope-tagged constructs of TGN38 or
furin (7, 25). Cells were fixed in methanol/acetone (1:1, v/v) for 10 min at Identification of
The other novel protein was named
Northern analyses revealed expression of human Detection of Endogenous
To gain further insight into the physical properties and composition of
the Identification of the Other Subunits of AP-4--
We hypothesized
that the polypeptides associated with
To test these predictions, we raised antibodies to these putative AP-4
subunits and used these antibodies in immunoprecipitation-recapture experiments. AP-4 was first isolated from
[35S]methionine-labeled HeLa cells by immunoprecipitation
with the anti- Intracellular Localization of the AP-4 Complex--
The
distribution of the endogenous AP-4 complex within HeLa cells was
examined by immunofluorescence microscopy using the anti-
The distribution of AP-4 was next compared with those of the previously
described AP complexes by double immunofluorescence staining. AP-1 is
mainly associated with the TGN (4), although it has also been detected
on peripheral cytoplasmic foci corresponding to endosomes (30, 31)
(Fig. 7E). AP-2 is normally associated with coated pits at
the plasma membrane (4) (Fig. 7H). The localization of AP-3
has not yet been established with certainty, although recent evidence
suggests that it is associated with a peripheral sorting compartment
related to endosomes (7, 11) (Fig. 7K). The distribution of
AP-4 most closely matched that of AP-1 in the juxtanuclear area (Fig.
7, D-F). A detailed inspection, however, revealed that the
staining patterns for AP-4 and AP-1 were not completely overlapping
(Fig. 7, D-F, insets), suggesting that the two
complexes may be associated with different regions of the juxtanuclear
structure. AP-4 exhibited little or no colocalization with AP-2 (Fig.
7, G-I) and AP-3 (Fig. 7, J-L).
The distribution of AP-4 was also compared with those of several
markers of compartments of the secretory and endocytic pathways. The
highest degree of colocalization was observed with the TGN markers
furin (Fig. 8, A-C) and TGN38
(Fig. 8, D-F). The AP-4 staining pattern also resembled to
some extent that of a 58-kDa cis-medial Golgi protein (Fig.
8, G and H). However, merging of the two images
(Fig. 8I) yielded much less overlap than that observed with
markers of the TGN. The distribution of AP-4 showed little colocalization with that of Lamp-1, the transferrin receptor, or the
cation-dependent mannose 6-phosphate receptor, all of which are considered markers for distinct compartments of the
endosomal-lysosomal system (data not shown). Taken together, the
immunofluorescence microscopy analyses suggested that AP-4 is
associated with the TGN or with a compartment that is immediately
adjacent to it.
In recent years, there have been major advances in our
understanding of the molecular machinery that mediates sorting of
integral membrane proteins in the secretory and endocytic pathways. Our laboratory has been particularly interested in a subset of protein coats that contain heterotetrameric adaptor complexes among their major
constituents. Three AP complexes have been described to date: AP-1,
AP-2, and AP-3 (Fig. 1A). In this study, we describe a
novel, widely expressed adaptor-like complex that we have named AP-4.
In keeping with the nomenclature of heterotetrameric adaptors, the four
subunits of AP-4 were named The complete primary structures of the The Messenger RNAs encoding the subunits of AP-4 were detected in all
tissues examined (this study and Ref. 18). While the expression patterns of the human µ4, The existence of both cytosolic and membrane-bound pools of AP-4
supports the notion that this complex is a component of a protein coat
that cycles between the cytosol and membranes, as is the case for other
coats. Cycling of AP-1, AP-3, and the non-clathrin COPI coat is
regulated by the small GTP-binding protein, ARF1, with the active
(GTP-bound) form of this protein promoting coat association to
membranes (19, 22, 35). The drug brefeldin A inhibits exchange of GDP
for GTP on ARF1 (29) and therefore causes redistribution of the coats
to the cytosol due to a blockage in membrane association. By
immunofluorescence microscopy, we observed a dramatic effect of the
drug on the subcellular localization of AP-4. Dispersal of AP-4 from a
juxtanuclear structure to the cytoplasm occurred within 30-60 s of
incubation of HeLa cells with the drug at 37 °C (Fig. 7C
and data not shown). At these short incubation times, the drug also
elicited complete redistribution of the Golgi-associated AP-1 and COPI
coats but did not affect the distribution of Golgi-associated integral
membrane proteins such as galactosyl transferase or TGN46.2
Although we cannot formally exclude the possibility that the observed
effect of the drug on AP-4 distribution is due to the formation of
small vesicles containing the complex, our results are consistent with
the idea that membrane association of AP-4 may be regulated by ARF1 or
a related protein.
Our immunofluorescence microscopy analyses suggest that AP-4 is
associated with the TGN or a compartment in the vicinity of the TGN.
AP-4 could thus be involved in one of a number of
vesicle-budding/sorting events that are thought to take place in
post-Golgi compartments. For example, the lysosomal membrane protein
Lamp-1, the cation-dependent mannose 6-phosphate receptor,
and invariant chain-containing class II molecules of the major
histocompatibility complex are all sorted to endosomal or lysosomal
compartments from the TGN (36-38). Some endocytic receptors, such as
the transferrin receptor and the asialoglycoprotein receptors, have
been shown to transit from the TGN to early endosomes prior to their
delivery to the cell surface (39, 40). The TGN is also thought to be
the site of formation of vesicles carrying cargo to the apical and
basolateral plasma membrane of polarized epithelial cells (41).
Finally, other sorting events could take place within structures
located in the proximity of the TGN, such as some endosomal
compartments. AP-4 could be solely responsible for one of these sorting
processes or could function as an alternative adaptor for processes
that are also mediated by AP-1 or AP-3. The availability of reagents for AP-4 should now allow experiments aimed at elucidating these issues.
and
subunits named
4 and
4, respectively. An
antibody to
4 recognized in human cells an ~83-kDa polypeptide
that exists in both soluble and membrane-associated forms. Gel
filtration, sedimentation velocity, and immunoprecipitation experiments
revealed that
4 is a component of a multisubunit complex (AP-4) that
also contains the
4 polypeptide and two additional adaptor subunit
homologs named µ4 (µ-ARP2) and
. Immunofluorescence analyses
showed that AP-4 is associated with the trans-Golgi network
or an adjacent structure and that this association is sensitive to the
drug brefeldin A. We propose that, like the related AP-1, AP-2, and
AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of
integral membrane proteins in mammalian cells.
INTRODUCTION
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Abstract
Introduction
References
/
/
and
1/
2/
3, ~90-130 kDa), one
medium chain (µ1/µ2/µ3, ~50 kDa), and one small chain
(
1/
2/
3, ~20 kDa) (Fig. 1A). The analogous
subunits of the complexes are all structurally related and probably
fulfill similar functions. For instance, the µ chains of the three
complexes are involved in the recognition of tyrosine-based signals
(5-7), and the
chains interact with clathrin (8-11). The
functions of the
/
/
and the
subunits are less clear,
although AP-2
has been shown to interact with other components of
the protein sorting machinery such as amphiphysin, dynamin, Eps15, and
epsin (12-14).
and
4), a medium chain
(µ-ARP2, herein referred to as µ4), and a small chain (
4) (Fig.
1B). AP-4 is widely expressed in mammalian tissues and
associates with membranes as a peripheral membrane protein. By
immunofluorescence microscopy, AP-4 was found to localize to the TGN or
a neighboring compartment and to be sensitive to brefeldin A, a drug
that also affects the distribution of AP-1 and AP-3 (7, 19, 20). These
properties of AP-4 are similar to those of other AP complexes,
suggesting that AP-4 may be a novel component of the cellular machinery
that sorts integral membrane proteins in mammalian cells.
EXPERIMENTAL PROCEDURES
4 and
4 cDNAs--
All EST clones used
in this study were from the I.M.A.G.E. Consortium (Lawrence Livermore
National Laboratory, Livermore, CA) and were purchased from the
American Type Culture Collection (Rockville, MD). DNA sequencing was
performed on both strands using the dideoxy method. The mouse EST clone
619775 consisted of a portion of the
4 cDNA spanning nucleotide
656 to the start of the poly(A) tail, while the mouse EST clones 442436 and 572356 comprised the complete coding region of
4 together with
both the 5'- and 3'-untranslated regions. Human EST clones 51094 and 206232 consisted of 3' fragments of the
4 cDNA starting at
nucleotides 1414 and 1724, respectively. The missing 5' portion of the
4 cDNA was obtained from a human skeletal muscle Marathon
ReadyTM cDNA library (CLONTECH, Palo Alto, CA)
by sequential 5'-RACE PCR using primers complementary to nucleotides
1657-1683 and 1626-1651 for the first and second (nested) PCR steps,
respectively. The final PCR product was cloned into the pNoTA/T7 vector
(5 Prime
3 Prime, Boulder, CO), and several independent clones were
isolated and sequenced.
4 and
4 probes were obtained by PCR. The
4 probe comprised nucleotides 1776-2226 of the full-length
4
cDNA, while the probe for
4 consisted of its complete coding sequence.
4C fusion
construct was generated by PCR amplification of a cDNA segment
encoding residues 601-739 of human
4, followed by cloning in frame
into the BamHI-NotI sites of the pGEX-5X-1 vector (Pharmacia Biotech, Uppsala, Sweden). The GST-
4-(18-54) and
GST-µ4-(122-326) constructs, bearing residues 18-54 of mouse
4
and residues 122-326 of human µ4/µ-ARP2 (EMBL data bank accession no. Y08387), respectively, were prepared by PCR and subsequent cloning
into the EcoRI-NotI sites of the pGEX-5X-1
vector. The constructs were verified by DNA sequencing. Overexpression
of GST-
4C and GST-
4-(18-54) in Escherichia
coli yielded soluble proteins that were affinity-purified on
glutathione-Sepharose 4B beads (Pharmacia Biotech) according to the
manufacturer's instructions. The GST-µ4-(122-326) fusion protein
was insoluble and was purified from inclusion bodies by preparative
SDS-PAGE.
4 and
4 were
raised in rabbits by immunization with purified GST-
4C
and GST-
4-(18-54) fusion proteins, respectively. The antibodies
were affinity-purified (7) and subsequently absorbed with immobilized
GST (Pierce). An antiserum to µ4 was generated by immunizing rabbits
with the GST-µ4-(122-326) fusion protein that had been purified on
polyacrylamide gels. The anti-
antiserum was raised in rabbits using
as the immunogen the peptide TALTSKHEEEKLIQQELSSL (Zymed
Laboratories Inc., San Francisco, CA). The anti-AP-3 mouse
antiserum was generated by immunization with a mixture of GST fusion
proteins bearing portions of all of the AP-3 subunits; preparation of
these GST-fusion proteins is described elsewhere (7, 21, 22).
Preparation of rabbit polyclonal antibodies to the AP-3
3 subunit
and to GST has been described previously (7, 11). The 58K-9 monoclonal antibody to the Golgi 58-kDa protein was purchased from Sigma. The
sources of the remaining antibodies used in this study are indicated
elsewhere (7, 17).
20 °C and subsequently air-dried. Incubation with the
primary antibody diluted in PBS, 0.1% (w/v) saponin, 1 g/liter BSA, 50 mg/liter GST was carried out for 1 h at room temperature. Unbound antibodies were removed by rinsing with PBS for 5 min, and cells were
subsequently incubated with secondary antibodies (Cy3-conjugated donkey
anti-rabbit Ig or Alexa 448-conjugated donkey anti-mouse Ig) diluted in
PBS, 0.1% (w/v) saponin, 1 g/liter BSA, for 30-60 min at room
temperature. After a final rinse with PBS, coverslips were mounted onto
glass slides with Fluoromount G (Southern Biotechnology Associates,
Birmingham, AL). Fluorescence images were acquired on a Zeiss LSM 410 confocal microscope (Carl Zeiss Inc., Thornwood, NY).
RESULTS
4 and
4--
Complementary DNAs encoding
two novel mammalian proteins related to subunits of the AP-1, AP-2, and
AP-3 complexes were identified through BLAST searches of EST data
bases. One of the proteins was named
4 on the basis of its homology
to the AP
subunits. Human
4 was predicted to be a protein of 739 amino acids and a molecular mass of 83,262 Da. Homology of
4 to the
other AP
subunits was restricted to the so-called "trunk"
amino-terminal domain, with the first ~580 residues of
4 sharing
28% identical amino acids with the trunk domain of mammalian
1 and
2, and 21% identical amino acids with the corresponding segment in
human
3A and
3B (Fig. 2A and data not shown). In these
molecules, the trunk domain is linked to a carboxyl-terminal "ear"
domain by a flexible, solvent-accessible "hinge" region (26).
Interestingly, secondary structure and accessibility predictions (27,
28) revealed that residues ~580-600 of
4 would correspond to a
solvent-accessible, random coil structure preceding a carboxyl-terminal
region with a high
-helix content (not shown). It is therefore
plausible that the
4 molecule has a domain organization similar to
that of the other AP
subunits, albeit with smaller hinge and ear domains (Fig. 1B).
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Fig. 1.
Schematic depiction of the subunit
composition and possible domain organization of the AP-1, AP-2, and
AP-3 complexes (A) and the related AP-4 complex
described in this study (B). Each complex
consists of four different subunits, the analogous subunits of the
complexes displaying significant homology to one other at the amino
acid level. The complexes are believed to adopt a mouse head-like
structure with a "head," comprising the µ and subunits and
the amino-terminal "trunk" domain (T) of the two large
subunits, and two "ears" (E) that correspond to the
carboxyl-terminal domains of the large subunits and are linked to the
head by flexible "hinges" (H). Only a portion of the
primary structure of
has been determined to date; its putative
domain organization has been modeled after those of its counterparts in
the AP-1, AP-2, and AP-3 complexes.
4 based on its homology to
previously known AP
subunits (Fig.
2B). The mouse
4 cDNA encodes a 144-residue protein with a calculated molecular mass of
16,818 Da. Homology of
4 to the other AP
polypeptides was significant throughout its entire amino acid sequence (37-43% identity; Fig. 2B and data not shown).
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Fig. 2.
Sequence alignments of
4 and
4 with selected
members of the AP
and AP
protein families. Sequences were aligned using the PILEUP
program. A, comparison of the predicted amino acid sequence
of human
4 to residues 1-577 (trunk domain) of rat
2
(GenBankTM accession no. M77245) and to residues 1-642
(trunk/A domain) of human
3A (GenBankTM accession no.
U81504). Amino acid residues conserved in at least two of the three
sequences are highlighted on a black
background. B, alignment of the primary structure
of mouse
4 with those of mouse
1A (GenBankTM
accession no. M62418), rat
2 (GenBankTM accession no.
M37194), and human
3A (EMBL Nucleotide Data Base accession no.
X99458). Residues conserved in at least three of the four sequences are
highlighted.
4 and mouse
4
mRNA species in all tissues examined, although some differences in
the expression patterns were observed (Fig.
3). The sizes of the major ~2.5-kb and
~1.2-1.4-kb species detected with the
4 and
4 probes were
consistent with those of the isolated
4 and
4 cDNAs,
respectively. A minor ~6-kb species was also detected in some human
tissues with the
4 probe; the nature of this species is unknown,
although it could correspond to an unprocessed or alternatively spliced
form of the
4 mRNA.
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Fig. 3.
Northern blot analysis of the expression
of 4 (A) and
4 (B) mRNAs in normal
tissues. Multiple tissue blots were incubated with specific
32P-labeled probes as described elsewhere (7). The
positions of molecular size markers (in kb) are indicated on the
left. Notice the presence of a ~2.5-kb band corresponding
to the full-length, mature
4 mRNA in all lanes in A
and that of a ~1.2-1.4-kb band corresponding to the
4 mRNA in
all lanes in B.
4 with a Specific Antibody--
To
characterize the
4 protein, we raised a polyclonal antibody to its
carboxyl-terminal region (anti-
4C). This antibody immunoprecipitated under nondenaturing conditions several proteins from
detergent lysates of [35S]methionine-labeled cells (Fig.
4A). In order to identify
unambiguously the endogenous
4 protein, the immunoprecipitate was
denatured with SDS and then subjected to a second immunoprecipitation
step using the same antibody. This immunoprecipitation-recapture
procedure (7, 23) resulted in the isolation of a single
35S-labeled polypeptide that migrated on SDS-PAGE with an
apparent molecular mass of ~83-kDa (Fig. 4B,
lane 1), a value that was in close agreement with
the calculated molecular mass of
4. The specificity of the
immunoprecipitation was corroborated by competition with excess
GST-
4C (Fig. 4B, lane
2) and by using an irrelevant antibody (to BSA) in the
recapture step (Fig. 4B, lane 3). The same
immunoprecipitation-recapture procedure was used to establish that
4
was present in both cytosolic (C) and postnuclear membrane (M) fractions (Fig. 4C). The relative yields of
soluble and membrane-associated forms of
4 were influenced by the
composition of the homogenization buffer (Fig. 4C), as
previously observed for the
3 subunit of AP-3 (7). In addition, the
membrane-associated form could be partially extracted with either 0.5 M Tris-HCl (Fig. 4D) or 1 M NaCl
(data not shown), thus suggesting that
4 associated with membranes
as a peripheral membrane protein.
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Fig. 4.
Detection and partial membrane association of
endogenous 4 from HeLa cells.
A, a Triton X-100 extract prepared from HeLa cells
metabolically labeled with [35S]methionine was subjected
to immunoprecipitation (IP) using an antibody to
4 or an
irrelevant control (c) antibody. Immunoprecipitates were analyzed by
SDS-PAGE and fluorography. The arrow points to an ~83-kDa
protein that was present in the immunoprecipitate obtained with the
anti-
4C antibody and was absent in the control
immunoprecipitate. B, isolation of endogenous
4 by
immunoprecipitation-recapture (23). The immunoprecipitate obtained with
the anti-
4C antibody was denatured in the presence of
SDS, diluted, and then subjected to a second immunoprecipitation step
using either the same specific antibody (
4,
lanes 1 and 2) or and irrelevant control antibody
(c, lane 3). The resulting samples were analyzed
by SDS-PAGE and fluorography. Notice the presence of the ~83-kDa
protein band that was recognized by the antibody to
4 in the
presence of GST but not in the presence of the competing antigen
(GST-
4C). C, immunoprecipitation of
4 from
cytosolic (C) and postnuclear membrane (M)
fractions that were obtained by ultracentrifugation of
[35S]methionine-labeled HeLa cell extracts containing
either 0.25 M sucrose or 0.15 M KCl (7).
D, immunoprecipitation of
4 from supernatants
(S) or pellets (P) obtained by
ultracentrifugation of membranes that had been incubated overnight at
4 °C with a low salt buffer (7) containing no additive (
) or 0.5 M Tris-HCl (+Tris).
4 Is a Component of a Large Complex--
Upon fractionation of
human fibroblast cytosol by gel filtration under nondenaturing
conditions,
4 could be detected by immunoblotting in fractions
corresponding to a Stokes radius of ~65 Å (Fig.
5A). This size was
significantly larger than that predicted for monomeric
4 and only
slightly smaller than those of the AP-1, AP-2, and AP-3 complexes (Fig.
5A), thus suggesting that endogenous
4 was part of a
complex.
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Fig. 5.
Association of endogenous
4 into a complex. A, Superose 6 gel
filtration analysis of a cytosolic fraction from unlabeled M1
fibroblasts (7). Eluted fractions were analyzed by immunoblotting using
antibodies to AP-1
(100/3), AP-2
(100/2), AP-3
3, and
4.
The elution positions of molecular size markers (Stokes radii given in
Ångstroms) are indicated by the arrows. B,
fractionation of a cytosolic preparation from
[35S]methionine-labeled HeLa cells by velocity
sedimentation on a 5-20% (w/v) sucrose gradient. Fractions were
immunoprecipitated (IP) with an irrelevant control antibody
(to GST) or with the anti-
4C antibody, and the resulting
precipitates were analyzed by SDS-PAGE and fluorography. Three
overlapping portions of the fluorograms, corresponding to different
exposure times, are shown. The migration of standard proteins on the
sucrose gradient (s20, W values given
in Svedberg units) are indicated. Notice the specific co-precipitation
of four polypeptides (labeled
,
4, µ4, and
4) by the
anti-
4C antibody in fractions 7 and 8 of the sucrose
gradient.
4-containing complex, we performed sedimentation analyses on a
sucrose gradient of a cytosolic extract from
[35S]methionine-labeled HeLa cells. Gradient fractions
were subjected to immunoprecipitation with anti-
4 and irrelevant
antibodies. Using this approach, we observed co-immunoprecipitation of
the ~83-kDa protein (corresponding to endogenous
4) with three
other polypeptides of apparent molecular masses ~140, ~50, and
~17 kDa (Fig. 5B). These polypeptides were not detected in
the control immunoprecipitation with the irrelevant antibody (to GST)
and were found to co-sediment with
4 on the sucrose gradient (Fig. 5B, fractions 7 and 8). The
position of
4 and its associated polypeptides on the gradient
corresponded to a species with a sedimentation coefficient of ~10 S. Using this value and the estimated Stokes radius (see above), we
calculated a molecular mass for the
4-containing complex of 280 ± 15 kDa. This value fits within the experimental error the predicted
molecular mass of a complex containing one molecule each of
4 and
the three associated polypeptides (i.e. ~290 kDa). Taken
together, these results indicated that
4 is a component of a
multisubunit protein complex, which we named AP-4 based on its
structural similarity to the previously characterized AP-1, AP-2, and
AP-3 adaptors.
4 could correspond to other
homologs of known AP subunits. Likely candidates for the ~17- and
~50-kDa polypeptides were the
4 protein identified in this study
and the previously described AP µ subunit homolog µ4/µ-ARP2 (18).
In addition, a search of EST data bases resulted in the identification
of a human cDNA clone (I.M.A.G.E. clone ID 1031294) containing a
~0.4-kb insert that encodes a novel protein sequence with significant
homology to the trunk region of the AP subunits
,
, and
(25-30% identical amino acids over a 132-residue overlap; data not
shown). Northern blot analysis indicated that this cDNA fragment
corresponded to a ubiquitously expressed mRNA species of about 7 kb
(data not shown). Based on this information, we hypothesized that this
as yet uncharacterized protein, herein referred to as
, could
correspond to the ~140-kDa subunit of the AP-4 complex.
4C antibody, and the immunoprecipitate was
then denatured and subjected to reprecipitation with the same antibody
or with antibodies to the other putative subunits (Fig.
6). As mentioned above, reprecipitation
with the anti-
4C antibody yielded the ~83-kDa
polypeptide (Fig. 6, lane 8). In addition, reprecipitation with antibodies to
4, µ4, and
resulted in the isolation of the
~17-, ~50-, and ~140-kDa subunits of the complex, respectively (Fig. 6). Specificity was verified by control reprecipitations using
irrelevant rabbit immunoglobulins or preimmune sera (Fig. 6) or by
competition experiments using purified antigens (data not shown).
Furthermore, none of our antibodies to AP-4 subunits recognized
components of AP-1, AP-2, or AP-3 on similar
immunoprecipitation-recapture experiments (data not shown).
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Fig. 6.
Identification of the subunits of AP-4 by
immunoprecipitation-recapture. The AP-4 complex was
immunoprecipitated from [35S]methionine-labeled HeLa
cells by using the anti- 4C antibody (Ab). The
immunoprecipitated material was denatured by heating at 95 °C in the
presence of SDS and dithiothreitol, diluted 20-fold, and subsequently
subjected to a second immunoprecipitation step using specific
antibodies to
4, µ4,
, and
4, as well as preimmune sera
(lanes 3 and 5) or irrelevant rabbit Ig (lanes 1 and 7) as nonspecific controls (c).
4C antibody. AP-4 was localized to a punctate
juxtanuclear structure reminiscent of the Golgi complex (Fig.
7, B, D,
G, and J). Staining was specific as judged by its
competition with an excess of the GST-
4C fusion protein
(Fig. 7A) but not GST (Fig. 7B). In addition,
treatment of the cells with brefeldin A, a drug that inactivates the
small GTP-binding protein ARF1 (29), caused a rapid dispersal of AP-4
into the cytoplasm (Fig. 7C).
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Fig. 7.
Immunofluorescence analysis of the
localization of AP-4 and other AP complexes in HeLa cells. Fixed
cells were incubated with the rabbit anti- 4C antibody in
the presence of either GST-
4C (A) or GST
(B-L) and with mouse antibodies to either AP-1 (100/3;
E and F, green), AP-2 (AP.6;
H and I, green), or AP-3 (K and
L, green) and subsequently with Cy3-conjugated
antibodies to rabbit Ig and Alexa448-conjugated antibodies to mouse Ig.
C, cells were incubated for 1 min at 37 °C in the
presence of 5 mg/liter brefeldin A prior to fixation. Bar,
10 µm.
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Fig. 8.
Double immunofluorescence staining of AP-4
and Golgi markers. HeLa cells transfected with HA epitope-tagged
constructs of furin (A-C) and TGN38 (D-F), as
well as untransfected cells (G-I), were fixed with
methanol/acetone and incubated simultaneously with the rabbit
anti- 4C antibody (A, C,
D, F, G, and I;
red) and with monoclonal antibodies to either the HA epitope
(B, C, E, and F;
green) or to the Golgi 58-kDa protein (H and
I; green). Bound antibodies were revealed with
Cy3-conjugated anti-rabbit Ig and Alexa448-conjugated anti-mouse Ig.
Bar, 10 µm.
DISCUSSION
,
4, µ4, and
4 (Fig.
1B). All of the AP-4 subunits are structurally related to
their counterparts in other AP complexes, suggesting that AP-4 may
resemble the other complexes with regard to overall conformation and
role in protein sorting.
4 and
4 subunits of AP-4
are reported in this study (Fig. 2). Unlike
4, which displays a
significant degree of sequence similarity to other AP
chains throughout the entire molecule,
4 homology to other AP
subunits is restricted to the amino-terminal trunk domain. This domain is
thought to mediate association with the other subunits of the AP
complexes (32, 33). On the other hand, the hinge-ear regions of
1,
2, and
3 (A or B) have been shown to interact with clathrin (10,
11, 34). A potential clathrin-binding motif, consisting of bulky
hydrophobic and acidic residues, has recently been identified in the
hinge-ear regions of
1,
2, and
3 (11). Interestingly, although
4 seems to bear hinge- and ear-like domains, these regions are
devoid of such a motif. This is consistent with the observations that
AP-4 was not enriched in a preparation of clathrin-coated vesicles from
bovine brain and that a GST-
4C fusion protein failed to
bind clathrin under conditions in which similar GST-
2 or -
3A fusion proteins displayed significant clathrin binding
activity.2 Since these
results are negative, however, further work will be required to
establish whether in vivo AP-4 interacts with clathrin or is
a component of a non-clathrin coat.
subunit of AP-4 was identified by immunoprecipitation-recapture
as the product of a novel cDNA for which we have obtained only a
partial sequence. Similar experiments also allowed us to identify the
µ4 subunit of AP-4 as a protein previously named µ-ARP2 (18). This
protein belongs to the family of related AP µ subunits that have been
shown to interact with tyrosine-based sorting signals (5-7). It is
therefore tempting to speculate that µ4, and by extension the whole
AP-4 complex, could play a role in sorting events mediated by
tyrosine-based signals.
4, and
are similar to each other, they differ somewhat from that of mouse
4 (Fig. 3, data not shown, and Ref. 18). The significance of these differences is at present unclear, since mRNA levels do not necessarily correlate with
protein levels, especially in the case of multisubunit complexes.
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ACKNOWLEDGEMENTS |
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We thank Marie-Christine Fournier for excellent technical assistance and Chean Eng Ooi for critical reading of the manuscript.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF092093 and AF092094.
Supported by a National Research Council Research Associateship.
§ To whom correspondence should be addressed: CBMB, NICHD, National Institutes of Health, Bldg. 18T, Rm. 101, Bethesda, MD 20892. Tel.: 301-496-6368; Fax: 301-402-0078; E-mail: juan{at}helix.nih.gov.
2 E. C. Dell'Angelica, and J. S. Bonifacino, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AP, adaptor protein; BSA, bovine serum albumin; EST, expressed sequence tag; GST, glutathione S-transferase; kb, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; TGN, trans-Golgi network.
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REFERENCES |
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