(Received for publication, February 24, 1997, and in revised form, April 18, 1997)
From the Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892
Recent studies have described a widely expressed
adaptor-like complex, named AP-3, which is likely involved in protein
sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of
the subunits of this complex, which we call 3A-adaptin. The
predicted amino acid sequence of
3A-adaptin reveals that the protein
is closely related to the neuron-specific protein
-NAP (61% overall
identity) and more distantly related to the
1- and
2-adaptin
subunits of the clathrin-associated adaptor complexes AP-1 and AP-2,
respectively. Sequence comparisons also suggest that
3A-adaptin has
a domain organization similar to
-NAP and to
1- and
2-adaptins.
3A-adaptin is expressed in all tissues and cells
examined. Co-purification and co-precipitation analyses demonstrate
that
3A-adaptin corresponds to the ~140-kDa subunit of the
ubiquitous AP-3 complex, the other subunits being
-adaptin, p47A
(now called µ3A) and
3 (A or B).
3A-adaptin is phosphorylated
on serine residues in vivo while the other subunits of the
complex are not detectably phosphorylated.
3A-adaptin is not present
in significant amounts in clathrin-coated vesicles. The characteristics
of
3A-adaptin reported here lend support to the idea that AP-3 is a
structural and functional homolog of the clathrin-associated adaptors
AP-1 and AP-2.
Cytosolic protein coats function as mediators of vesicle budding
and selection of cargo molecules at different stages of the secretory
and endocytic pathways (reviewed in Refs. 1 and 2). Among the various
protein coats that have been described to date, those containing the
protein clathrin are the most extensively characterized (reviewed in
Refs. 3-5). Clathrin coats are found in association with the cytosolic
aspect of the trans-Golgi network and the plasma membrane,
and with coated vesicles that originate from these organelles. In
addition to clathrin, these coats contain specific protein complexes
known as adaptors or APs. One of these adaptors, AP-1, is a component
of trans-Golgi network clathrin coats and consists of four
subunits: - and
1-adaptins (~100 kDa), µ1 (~47 kDa), and
1 (~19 kDa). Another adaptor, AP-2, is associated with plasma
membrane clathrin coats and is also composed of four subunits:
- and
2-adaptins (~100 kDa), µ2 (~50 kDa), and
2 (~17 kDa). The
analogous subunits of AP-1 and AP-2 display significant homology to
each other; in addition, the adaptor complexes themselves exhibit a
similar overall structure of a "head" with two protruding
"ears," each separated from the head by a flexible "hinge"
(3-5, 46).
A major function of AP-1 and AP-2 is to link clathrin lattices to the
corresponding membranes. This role is fulfilled by the - and
1-adaptin subunits of AP-1 and the
- and
2-adaptin subunits of
AP-2 (6-9). The adaptors are also responsible for the recognition of
sorting signals present in the cytosolic domains of integral membrane
proteins (10-21), an event that leads to the concentration of these
proteins within clathrin-coated areas of the trans-Golgi network and the plasma membrane. Recent evidence suggests that the µ1
subunit of AP-1 and the µ2 subunit of AP-2 are directly involved in
signal recognition (16, 18, 22).
In the past few years, it has become clear that the structure and
function of clathrin coats serve as paradigms for other protein coats.
Indeed, some subunits of the non-clathrin coat, COPI, are structurally
related to subunits of AP-1 and AP-2 (23-26). In addition, recent
studies have identified other mammalian proteins that display
significant homology to AP-1 and AP-2 subunits and are components of
previously unknown coats. Pevsner et al. (27) isolated
cDNAs encoding two proteins, named p47A and p47B, that exhibit
~80% identity to each other and ~30% identity to µ1 and µ2.
Newman et al. (28) described another protein, named -NAP, that is ~30% identical to
1- and
2-adaptins. Finally, Watanabe et al. (29) and Dell'Angelica et al. (30)
reported the identification of two proteins named
3A and
3B;
these proteins are 84% identical to each other and ~30% identical
to
1 and
2. Some of these subunits are expressed in a wide
variety of tissues and cell lines (p47A,
3A, and
3B), while
others are only expressed in brain, spinal cord, and neuronal cell
lines (p47B and
-NAP).
Not surprisingly, these homologs of AP-1 and AP-2 subunits were found
to exist as heterotetrameric assemblies resembling adaptor complexes
(30, 31). One of these complexes, expressed in neuronal cells, contains
-NAP and either p47A or p47B (31). A similar complex, which was
named AP-3, is expressed in all cells examined to date and contains
subunits of ~160, ~140, ~47, and ~22 kDa (30). The ~47-kDa
protein corresponds to p47A while the ~22-kDa protein is either
3A
or
3B. The ~140-kDa subunit of this complex is immunologically
related to the neuronal protein
-NAP but distinct from it based on
its expression in various non-neuronal cell lines (30).
In this paper we report the cloning of a novel cDNA encoding the
~140-kDa subunit of the ubiquitous AP-3 complex. This protein, referred to as 3A-adaptin, is closely related to
-NAP and
displays significant homology to
1- and
2-adaptins. Consistent
with it being a subunit of the ubiquitous AP-3 complex,
3A-adaptin
is expressed in a wide variety of tissues and cell lines. Biochemical analyses show that
3A-adaptin is phosphorylated on serine residues in vivo and is absent from clathrin-coated vesicles. The
similarity of
3A-adaptin to
1- and
2-adaptins lends support to
the idea that AP-3 is structurally and functionally related to AP-1 and AP-2, and that it is likewise involved in the regulation of
intracellular protein trafficking.
Two EST clones (GenBank
accession codes R02669[GenBank] and T98538[GenBank]) from human fetal liver/spleen
(Washington University-Merck EST Project) were found to encode portions
of a novel protein with significant homology to -NAP. Based on these
partial sequences, a full-length cDNA encoding this protein, named
3A-adaptin, was isolated from a Marathon-ReadyTM human pancreas
cDNA library (CLONTECH, Palo Alto, CA) by a
combination of 5
- and 3
-RACE1 PCR, using
the AdvantageTM cDNA PCR kit (CLONTECH). The specific primers used
in a first and second (nested) 5
-RACE reactions were complementary to
nucleotides 3181-3205 and 2955-2981 of the
3A-adaptin cDNA,
respectively. The first and second (nested) primers used for 3
-RACE
PCR corresponded to nucleotides 1773-1800 and 1898-1922 of the
full-length cDNA, respectively. Both 5
and 3
nested PCR products
were cloned into the pNoTA/T7 shuttle vector (5 Prime
3 Prime,
Inc., Boulder, CO). Several independent clones were isolated and
sequenced to guard against errors introduced by the DNA polymerase
during PCR amplification. Both strands were sequenced by the dideoxy
method.
The sources and culture conditions for all the human cell lines used in this study are described elsewhere (30).
Northern Blot and RT-PCR Analyses of mRNA ExpressionNorthern blot analysis was carried out as described
before (30). The 3A-adaptin probe was obtained by PCR using a 5
primer corresponding to nucleotides 1898-1922 and a 3
primer
complementary to nucleotides 2955-2981 of the full-length cDNA,
respectively. The probe used for detecting the
-NAP mRNA
consisted of a 450-base pair fragment which was PCR-amplified from the
EST clone 165789 (Washington University-Merck EST Project) using 5
and
3
primers corresponding to nucleotides 2000-2028 and 2424-2449 of
the full-length
-NAP cDNA (GenBank accession number: U37673[GenBank]),
respectively. The above sets of primers were also used for RT-PCR
analysis of the expression of
3A-adaptin and
-NAP mRNAs in
cell lines; the analysis was performed by using the Gene Amp® XL RNA
PCR kit (Perkin-Elmer, Branchburg, NJ) according to the manufacturer's
instructions.
To prepare a series of
GST fusion proteins bearing different segments of 3A-adaptin, the
corresponding cDNA fragments were engineered by PCR to be cloned
in-frame into the pGEX-5X-1 vector (Pharmacia Biotech, Uppsala,
Sweden). The
3A-adaptin cDNA segments corresponding to amino
acids 1-287, 1-642, and 810-1094 were cloned into the
EcoRI-NotI cloning sites of the vector, while the
segment encoding amino acids 643-809 was cloned into the
BamHI-NotI sites of the vector. The DNA sequence
of all the constructs was confirmed by manual sequencing. Fusion
proteins were expressed in Escherichia coli cells and then
affinity-purified using glutathione-Sepharose 4B beads (Pharmacia
Biotech Inc.) following the manufacturer's instructions.
Monoclonal anti--adaptin antibody 100/2 was
obtained from Sigma. The preparation and purification of polyclonal
rabbit antibodies to p47 (A and B) and
3 (A and B) were described
previously (30). The preparation of an antiserum against a GST fusion
protein that bears the hinge region of
-NAP
(GST-
-NAP647-796) has also been described previously
(30). Here, this antiserum was passed through a column containing GST
coupled to Affi-Gel 15 (Bio-Rad), to remove anti-GST antibodies, and
then affinity-purified (32) using as a ligand a peptide comprising
residues 647-796 of
-NAP. The purified antibody, herein referred to
as
3H1, recognizes the hinge region of both
-NAP and
3A-adaptin, as inferred from immunoprecipitation experiments with
the corresponding fragments translated in vitro. The
3H7
antibody was raised in rabbits by immunization with the
GST-
3A643-809 fusion protein. The antibody was
affinity-purified using as a ligand the same fusion protein coupled to
Affi-Gel-15 beads, and was subsequently immunoabsorbed with GST,
followed by GST-
-NAP647-796 to obtain a monospecific antibody to the hinge region of
3A-adaptin. The lack of
cross-reactivity between the
3H7 antibody and
-NAP was
corroborated by immunoprecipitation experiments and by immunoblotting.
The
3A1 and
3C1 antibodies were obtained by immunizing rabbits
with GST-
3A1-642 and GST-
3A810-1094
fusion proteins, followed by affinity purification on immobilized
GST-
3A1-287 and GST
3A810-1094,
respectively. Both antibodies were absorbed with GST. Polyclonal rabbit
antibodies to BSA (Cappel, Cochranville, PA) or to the FLAG epitope
(Santa Cruz Biotechnology, Santa Cruz, CA) were used as irrelevant
antibody controls. The preparation of an antibody to human
-adaptin
is described elsewhere.2
Metabolic labeling of M1 cells with [35S]methionine, immunoprecipitation-recapture experiments, and immunoblot analysis were performed as described previously (30).
Alkaline Phosphatase TreatmentThe subunits of the AP-3 complex were isolated from [35S]methionine-labeled M1 cells by immunoprecipitation-recapture (30). The immunoprecipitates were resuspended in 50 mM Tris-HCl (pH 8.5), 1 mM EDTA and divided into two aliquots. One of the aliquots was treated with 1 milliunit of calf intestinal alkaline phosphatase (Boehringer Mannheim) for 1 h at 37 °C; the other one was mock-treated. Samples were analyzed by SDS-PAGE (33) followed by fluorography.
Metabolic Labeling with [32P]Orthophosphate and Phosphoamino Acid AnalysisM1 cells were grown to almost
confluence in Dulbecco's modified Eagle's medium supplemented with
9% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 50 µg/ml gentamicin. Cells were detached from the
plates by treatment with 0.5 mg/ml trypsin, 10 mM EDTA in
phosphate-free Dulbecco's modified Eagle's medium, 25 mM
HEPES, collected by low-speed centrifugation, and then washed twice
with phosphate-free Dulbecco's modified Eagle's medium, 25 mM HEPES, 0.1% BSA. Subsequently, cells were suspended in
phosphate-free Dulbecco's modified Eagle's medium, 25 mM
HEPES, 0.1% BSA containing 2 mCi/ml [32P]orthophosphate
and incubated at 37 °C for 3 h. After the incubation, cells
were washed three times with ice-cold phosphate-buffered saline
containing 5 mM EDTA, 1 mM orthovanadate, and 1 mM sodium fluoride, and then lysed by incubation for 15 min
on ice with 1% (w/v) Triton X-100, 0.3 M NaCl, 50 mM Tris-HCl (pH 7.4), 10 mM iodoacetamide, 5 mM EDTA, 1 mM orthovanadate, 1 mM
sodium fluoride, 1 mM 4-(2-aminoethyl)-benzenesulfonyl
fluoride, 2 µg/ml leupeptin, 0.1% (w/v) BSA. Immunoprecipitation of
the AP-3 complex with an anti-3 antibody, dissociation of the
complex, and subsequent immunoprecipitation of the subunits with
specific antibodies were carried out as described previously (30).
Phosphoamino acid analysis of metabolically 32P-labeled
3A-adaptin was performed by two-dimensional thin layer electrophoresis (34).
The preparation of cytosol from
M1 cells and its fractionation by gel filtration were performed as
described previously (30). For isolation of the complex by affinity
chromatography, the cytosol was passed through a Protein A-Sepharose
column (1.6 ml) and then loaded onto a 0.4-ml column containing 0.7 mg
of purified anti-3 antibody which had been covalently coupled to
Protein A-Sepharose (32). Bound proteins were eluted with 0.1 M glycine (pH 2.5). A crude membrane fraction and purified
clathrin-coated vesicles from bovine brain were the kind gift of Lois
Greene and Evan Eisenberg (National Heart, Lung, and Blood Institute,
National Institutes of Health).
Our previous work demonstrated the existence in
non-neuronal cells of a protein immunologically related to the neuronal
-NAP (30). To identify this protein, we searched EST data bases for
-NAP homologs. Two EST clones from human fetal liver/spleen were found to encode a novel protein with significant homology to
-NAP. The complete cDNA was obtained using 5
- and 3
-RACE procedures based on partial sequences of the EST clones. Analysis of the sequence
of this 3950-base pair cDNA revealed a long open reading frame
encoding a protein of 1094 amino acids with a predicted molecular mass
of 121,350 Da. The protein displayed significant homology not only to
-NAP (Fig. 1), but also to the Saccharomyces cerevisiae chromosome VII open reading frame YGR261c and to
mammalian
1- and
2-adaptins (Fig. 2). The new
human protein was named
3A-adaptin.
Based on a hydropathy plot, 3A-adaptin can be divided into at least
three distinct regions: an amino-terminal (A) region
spanning residues 1-642, a strongly hydrophilic segment (H)
comprising residues 643-809, and a carboxyl-terminal (C)
region spanning residues 810-1094 (Fig. 2, A and
B). This structure is reminiscent of the domain organization
of other adaptins (3-5).
The homology of 3A-adaptin to
-NAP extends over the entire length
of their polypeptide chains (Fig. 1), although the degree of sequence
similarity varies in the three regions (Fig. 2B). The
percentage of conserved amino acid residues shared by the two proteins
is highest in the A region; this region also exhibits the
highest degree of similarity to the S. cerevisiae YGR261c
protein and to the mammalian
-adaptins (Fig. 2B). In the
1- and
2-adaptins, the amino-terminal segment corresponds to a
"head" or "core" domain of the proteins where interactions with
the other adaptor subunits are thought to take place. The head domains
of
1-adaptin,
2-adaptin, and
-NAP have been shown to contain
up to 14 Arm repeats (35, 36), a degenerate motif that
probably functions as a protein-protein interaction element. Analysis
of the
3A-adaptin sequence reveals that this protein also contains
12 or 13 Arm repeats in the A region (not shown).
The H region of 3A-adaptin is strongly hydrophilic and
rich in acidic residues (31%) and serine residues (26%) (Fig. 1).
This region contains many potential sites for phosphorylation by the
serine/threonine kinases casein kinase I and casein kinase II (21 and
25 sites, respectively; Refs. 37 and 38). The H region of
3A-adaptin is 35 and 19% identical to the analogous regions in
-NAP and YGR261c, respectively (Fig. 2B). Although the
percentage of identical residues is lower in this region, as compared
with the A region, the general characteristics of the
sequence (i.e. high content of acidic and serine residues) are conserved among these proteins. No significant homology to mammalian
-adaptins was observed in this region, although the analogous segment in
1- and
2-adaptins is also hydrophilic and displays a hinge-like structure.
Finally, the C region of 3A-adaptin is 50% identical to
the homologous segment of
-NAP but shows no significant homology to
1- and
2-adaptins and is absent from YGR261c (Fig.
2B). In the
-adaptins, this segment corresponds to the
"ear" or "appendage" domain.
A philogenetic tree constructed with the above sequences and that of
the more distantly related COPI subunit, -COP, (Fig. 2C)
shows that
3A-adaptin,
-NAP, and YGR261c all belong to a group
that may have diverged from the others early in the evolution of the
family. The mammalian
1 and
2-adaptins, a D. melanogaster
-adaptin and two other S. cerevisiae
adaptins (Sc ADB1 and Sc ADB2) cluster together in a separate branch.
The
-COPs from rat, fly, and yeast are distantly related to the
members of the other two groups. Thus, these sequence analyses define a
group of proteins (
3A-adaptin,
-NAP, and YGR261c) that are more
closely related to clathrin-associated
-adaptins than to COPI
components.
The
pattern of expression of the 3A-adaptin mRNA in various human
tissues and cell lines was examined by Northern blot analysis (Fig.
3). A single ~4.2-kb
3A-adaptin message was
detected in all tissues examined (Fig. 3A), as well as in
both non-neuronal (M1, HeLa, and RD4) and neuronal cell lines (H4,
SK-N-SH, SK-N-MC, and Ntera-2) (Fig. 3B). In contrast to the
3A-adaptin mRNA, the
-NAP mRNA is known to be expressed
only in brain (Ref. 28 and data not shown). Northern analyses of
various cell lines detected expression of the
-NAP mRNA only in
the neuronal precursor line Ntera-2 (Fig. 3B). Additional
analyses by RT-PCR, which is more sensitive than Northern analysis,
revealed the presence of small amounts of
-NAP mRNA in other
neuronal cells but not in non-neuronal cells (Fig. 3C).
Thus, the
3A-adaptin mRNA is widely expressed, whereas
expression of the
-NAP mRNA is restricted to brain and cell
lines of neuronal origin.
To
characterize the biochemical properties of the 3A-adaptin protein,
we raised antibodies to different regions of the molecule. The
antibodies were affinity-purified and used to identify the protein by
Western blot analysis of the human fibroblast cell line M1, which
expresses the
3A-adaptin mRNA but not the
-NAP mRNA (Fig.
3). Antibodies to both the H and C regions of
3A-adaptin (
3H7 and
3C1, respectively) were found to recognize a protein that migrated as a ~140-kDa polypeptide on SDS-PAGE and
that co-eluted on a gel filtration column with the ~22-kDa protein
3 (Fig. 4A), a known component of the AP-3
complex (30). The peak elution of
3A-adaptin and
3 corresponded
to a complex with Stokes radius of ~85 Å, as previously reported
(30).
To address directly the question of whether 3A-adaptin is a
component of AP-3, this complex was affinity-purified from M1 cells on
an anti-
3 column and the eluate was analyzed by Western blotting
with anti-
3 and anti-
3A-adaptin antibodies (Fig. 4B). The anti-
3 antibody recognized minor and major species of 20-22 kDa, as previously shown (30), whereas the different antibodies to
3A-adaptin recognized a ~140-kDa species (Fig. 4B).
Neither band was observed when the same procedure was performed on a
control column without anti-
3 antibody (data not shown). Thus,
3A-adaptin co-purifies with
3 on affinity chromatography.
The association of 3A-adaptin with
3 was also analyzed using an
immunoprecipitation-recapture technique (30). This technique consisted
of immunoprecipitating the AP-3 complex from
[35S]methionine-labeled M1 cells using an antibody to
3 and, after dissociation in the presence of SDS, isolating the
individual subunits of the complex by re-precipitation with specific
antibodies. Re-precipitation with the
3H1 antibody (Fig.
5A) and with the antibodies
3H7,
3A1,
and
3C1 (not shown) further confirmed that
3A-adaptin is indeed a
component of the AP-3 complex. Fig. 5A also illustrates
that, in addition to
3 (A or B) and
3A-adaptin, the other
components of AP-3 are p47A (27) and another protein known as
-adaptin.2
Because of the many
potential phosphorylation sites predicted from the sequence (see
above), we were interested in determining whether the protein was
phosphorylated in vivo. To this end, we treated
[35S]methionine-labeled 3A-adaptin isolated from the
cytosol of M1 cells with alkaline phosphatase and determined the
migration of the untreated and treated samples on SDS-PAGE. The
alkaline phosphatase treatment resulted in decreased migration of the
entire population of labeled
3A-adaptin molecules, as can be seen in Fig. 5A (lanes 5 and 6), and with
better resolution in Fig. 5B. This experiment suggested that
3A-adaptin is a phosphorylated protein at steady state. In contrast
to
3A-adaptin, none of the other subunits of AP-3 changed their
migration upon treatment with alkaline phosphatase (Fig.
5A).
The fact that the AP-3 complex exists both in soluble and
membrane-bound pools (30) led us to investigate whether phosphorylation of 3A-adaptin correlates with association to membranes. We found that both the cytosolic and membrane-bound forms of
3A-adaptin were
equally sensitive to alkaline phosphatase (Fig. 5B), thus deeming it unlikely that the observed phosphorylation regulates membrane association.
To confirm that 3A-adaptin is a phosphoprotein and to identify the
amino acids that are phosphorylated, M1 cells were metabolically labeled with [32P]orthophosphate and the AP-3 complex and
each of its subunits were isolated by immunoprecipitation with specific
antibodies (Fig. 6A). We observed that
3A-adaptin was the only subunit of the complex that incorporated
[32P]orthophosphate under the conditions of the
experiment (Fig. 6A). Phosphoamino acid analyses of
32P-labeled
3A-adaptin revealed that the phosphorylation
was on serine residues (Fig. 6B). From these experiments, we
concluded that
3A-adaptin exists as a serine-phosphorylated protein
under basal conditions. Although we did not detect phosphorylation of the other subunits of AP-3, it is possible that they are phosphorylated less extensively or that they are only phosphorylated under certain physiologic conditions.
AP-3 Is Not Enriched in Clathrin-coated Vesicles
Because of
the structural similarities of AP-3 to the clathrin-associated adaptors
AP-1 and AP-2, it was of interest to examine if AP-3 was associated
with clathrin-coated vesicles. To this end, we determined the amount of
3A-adaptin associated with bovine brain-coated vesicles in
comparison to a crude membrane fraction. This analysis was done by
Western blotting using a monospecific antibody (
3H7) to the
3A-adaptin hinge that does not recognize
-NAP. We performed a
similar analysis using antibodies to two other components of the AP-3
complex, p47 (A and B) and
3 (A and B), and to a component of AP-2,
-adaptin, as a control. Two forms of
-adaptin (
a
and
c; Ref. 39) were highly enriched in the
clathrin-coated vesicle preparation relative to the crude membrane
fraction (Fig. 7). This was in contrast to
3A-adaptin which was not detected in the clathrin-coated vesicles, although it was
present in high amounts in the crude membrane fraction (Fig. 7).
Similarly, p47 and
3 were not detected in the clathrin-coated vesicle fraction (Fig. 7). Thus, these experiments suggest that the
AP-3 complex is not associated with clathrin-coated vesicles.
In this study we describe a novel human protein named
3A-adaptin. The
3A-adaptin mRNA is expressed in a wide
variety of tissues and cell lines and the protein itself has been shown
to exist in various cell lines (this study; Ref. 30). Biochemical analyses demonstrate that
3A-adaptin is a subunit of the ubiquitous adaptor-like protein complex AP-3, which has been previously shown to
exist in association with trans-Golgi network and/or
endosomal compartments (30). The other subunits of the AP-3 complex are
-adaptin,2 p47A (Ref. 27; now called µ3A), and
3A
or
3B (29, 30) (Fig. 5). The AP-3 complex is not associated with
clathrin-coated vesicles, suggesting that it may be a component of a
different coat. In this regard, the ubiquitous AP-3 complex resembles
the complex containing the brain-specific
-NAP, which is also absent from clathrin-coated vesicles (28, 31).
The existence of a closely-related homolog of the brain-specific
-NAP was first evidenced by the immunoprecipitation of a ~140-kDa
protein from non-neuronal cells using an antibody to
-NAP (30).
Since
-NAP is not expressed in non-neuronal cells (Refs. 28 and 31,
this study), this protein had to be an immunologically cross-reactive
homolog. Sequence analyses now show that
3A-adaptin shares 61%
overall identity and 75% overall similarity with
-NAP.
-NAP
itself is part of a complex with either the brain-specific p47B (now
called µ3B) or the ubiquitous p47A (µ3A) subunits, and with two
other proteins that are also similar to subunits of AP-3 (31). This
suggests that both the ubiquitous
3A-adaptin-containing complex and
the brain-specific
-NAP-containing complex have a similar structure
and may even share some common subunits. Moreover, in cells that
express both the ubiquitous and brain-specific subunits (i.e. neurons), the subunits could combine to generate
several different complexes.
In addition to -NAP, other coat proteins display a lower but
significant degree of homology to
3A-adaptin. This includes the
S. cerevisiae gene product, YGR261c, also known as
APL6/YKS5. This protein is likely to be the yeast counterpart of
3A-adaptin and/or
-NAP and, in fact, has been shown to be part of
a complex with three other proteins (APL5/YKS4, APM3/YKS6, and
APS3/YKS7) which are closely related to AP-3
subunits.3
3A-adaptin also has a
significant degree of homology to the clathrin-associated adaptor
subunits
1- and
2-adaptins and to the COPI subunit
-COP. The
fact that
3A-adaptin is related to all of these organellar coat
proteins and the ability of p47A (µ3A) to bind tyrosine-based sorting
signals (30) strongly suggest that the AP-3 complex may be similarly
involved in the regulation of intracellular protein trafficking.
An examination of the 3A-adaptin sequence reveals at least three
distinct regions, named A (amino-terminal), H (hinge), and C (carboxyl-terminal) (Fig. 2B). The
three regions are likely analogous to structural and functional domains that have been well defined in
1- and
2-adaptins (40, 41). The
A region is homologous to the head or core domains of
1-
and
2-adaptins, which are involved in interactions with the other
subunits of AP-1 and AP-2 (9, 40, 42). The H region is
analogous to the hinge or "stalk" domains of
1- and
2-adaptins, which mediate interactions with clathrin (7, 43). Finally, the C region of
3A-adaptin might be analogous to
the ear or appendage domains of
1- and
2-adaptins. The function of the
1- and
2-adaptin ear domains is still unclear, although the analogous domain of
-adaptin has been shown to bind regulatory molecules such as dynamin (44) and Eps15 (45). The domain organization
of
-adaptin is also thought to resemble those of
- and
-adaptin.2 These similarities suggest that AP-3 may have
an overall structure analogous to AP-1 and AP-2.
A salient feature of 3A-adaptin is that it is phosphorylated on
serine residues (Figs. 5 and 6). Moreover,
3A-adaptin is the only
subunit of AP-3 that is detectably phosphorylated under the conditions
of our experiments. Although the sites of phosphorylation within
3A-adaptin have not been located, the presence of a large number of
consensus sequences for phosphorylation by casein kinase I and II in
the H region suggests that this region might be highly
phosphorylated. While the degree of identity of
3A-adaptin to
-NAP and S. cerevisiae YGR261c is lower in the
H region as compared with the A region, it is
noteworthy that the overall characteristics of the segment are
conserved among these proteins, including the presence of many
potential sites of phosphorylation by casein kinase I and II. Indeed,
-NAP is also heavily phosphorylated both in vivo and
in vitro (28) and YGR261c displays a genetic interaction
with casein kinase I in yeast cells.3 Components of the
clathrin-associated adaptors (47-50) and of COPI (51) have also been
shown to be phosphorylated, suggesting that phosphorylation might be a
common mechanism for regulating coat function within cells.
The findings presented here add to the growing evidence that AP-3 is a
structural and functional homolog of AP-1 and AP-2. Indeed, all three
complexes are capable of binding reversibly to membranes and have a
similar heterotetrameric structure. Moreover, the analogous subunits of
the three complexes are structurally related to each other and may even
exhibit a similar domain organization. Finally, the analogous subunits
might play similar roles. For instance, the p47A (µ3A) subunit of
AP-3 is capable of binding tyrosine-based sorting signals (22, 30),
like its relatives µ1 and µ2 (16, 18, 22). The comparable domain
organization of 1-,
2-, and
3A-adaptins suggests that they
might fulfill similar roles in the interaction of the adaptor complexes
with scaffolding proteins. Further studies of AP-3 should establish not
only the extent to which it resembles AP-1 and AP-2 but also the
functional differences that account for the existence of the three
complexes in all cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81504[GenBank].
We thank Kathryn O'Reilly and George Poy for their help with DNA sequencing, Evan Eisenberg and Lois Greene for their generous gift of reagents, the Washington University-Merck EST Project for generating the ESTs that made this project possible, and Jennifer Lippincott-Schwartz and Lawrence Samelson for review of the manuscript.