beta 3A-adaptin, a Subunit of the Adaptor-like Complex AP-3*

(Received for publication, February 24, 1997, and in revised form, April 18, 1997)

Esteban C. Dell'Angelica , Chean Eng Ooi and Juan S. Bonifacino Dagger

From the Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 beta 3A-adaptin. The predicted amino acid sequence of beta 3A-adaptin reveals that the protein is closely related to the neuron-specific protein beta -NAP (61% overall identity) and more distantly related to the beta 1- and beta 2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that beta 3A-adaptin has a domain organization similar to beta -NAP and to beta 1- and beta 2-adaptins. beta 3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that beta 3A-adaptin corresponds to the ~140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being delta -adaptin, p47A (now called µ3A) and sigma 3 (A or B). beta 3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. beta 3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of beta 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.


INTRODUCTION

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: gamma - and beta 1-adaptins (~100 kDa), µ1 (~47 kDa), and sigma 1 (~19 kDa). Another adaptor, AP-2, is associated with plasma membrane clathrin coats and is also composed of four subunits: alpha - and beta 2-adaptins (~100 kDa), µ2 (~50 kDa), and sigma 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 gamma - and beta 1-adaptin subunits of AP-1 and the alpha - and beta 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 beta -NAP, that is ~30% identical to beta 1- and beta 2-adaptins. Finally, Watanabe et al. (29) and Dell'Angelica et al. (30) reported the identification of two proteins named sigma 3A and sigma 3B; these proteins are 84% identical to each other and ~30% identical to sigma 1 and sigma 2. Some of these subunits are expressed in a wide variety of tissues and cell lines (p47A, sigma 3A, and sigma 3B), while others are only expressed in brain, spinal cord, and neuronal cell lines (p47B and beta -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 beta -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 sigma 3A or sigma 3B. The ~140-kDa subunit of this complex is immunologically related to the neuronal protein beta -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 beta 3A-adaptin, is closely related to beta -NAP and displays significant homology to beta 1- and beta 2-adaptins. Consistent with it being a subunit of the ubiquitous AP-3 complex, beta 3A-adaptin is expressed in a wide variety of tissues and cell lines. Biochemical analyses show that beta 3A-adaptin is phosphorylated on serine residues in vivo and is absent from clathrin-coated vesicles. The similarity of beta 3A-adaptin to beta 1- and beta 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.


EXPERIMENTAL PROCEDURES

Cloning of beta 3A-adaptin cDNA

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 beta -NAP. Based on these partial sequences, a full-length cDNA encoding this protein, named beta 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 beta 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 right-arrow 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.

Cells

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 Expression

Northern blot analysis was carried out as described before (30). The beta 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 beta -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 beta -NAP cDNA (GenBank accession number: U37673[GenBank]), respectively. The above sets of primers were also used for RT-PCR analysis of the expression of beta 3A-adaptin and beta -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.

Production of GST Fusion Proteins

To prepare a series of GST fusion proteins bearing different segments of beta 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 beta 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.

Antibodies

Monoclonal anti-alpha -adaptin antibody 100/2 was obtained from Sigma. The preparation and purification of polyclonal rabbit antibodies to p47 (A and B) and sigma 3 (A and B) were described previously (30). The preparation of an antiserum against a GST fusion protein that bears the hinge region of beta -NAP (GST-beta -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 beta -NAP. The purified antibody, herein referred to as beta 3H1, recognizes the hinge region of both beta -NAP and beta 3A-adaptin, as inferred from immunoprecipitation experiments with the corresponding fragments translated in vitro. The beta 3H7 antibody was raised in rabbits by immunization with the GST-beta 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-beta -NAP647-796 to obtain a monospecific antibody to the hinge region of beta 3A-adaptin. The lack of cross-reactivity between the beta 3H7 antibody and beta -NAP was corroborated by immunoprecipitation experiments and by immunoblotting. The beta 3A1 and beta 3C1 antibodies were obtained by immunizing rabbits with GST-beta 3A1-642 and GST-beta 3A810-1094 fusion proteins, followed by affinity purification on immobilized GST-beta 3A1-287 and GSTbeta 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 delta -adaptin is described elsewhere.2

Immunoprecipitation and Immunoblotting

Metabolic labeling of M1 cells with [35S]methionine, immunoprecipitation-recapture experiments, and immunoblot analysis were performed as described previously (30).

Alkaline Phosphatase Treatment

The 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 Analysis

M1 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-sigma 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 beta 3A-adaptin was performed by two-dimensional thin layer electrophoresis (34).

Other Materials and Methods

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-sigma 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).


RESULTS

Molecular Cloning and Sequence Analyses of a cDNA Encoding beta 3A-adaptin

Our previous work demonstrated the existence in non-neuronal cells of a protein immunologically related to the neuronal beta -NAP (30). To identify this protein, we searched EST data bases for beta -NAP homologs. Two EST clones from human fetal liver/spleen were found to encode a novel protein with significant homology to beta -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 beta -NAP (Fig. 1), but also to the Saccharomyces cerevisiae chromosome VII open reading frame YGR261c and to mammalian beta 1- and beta 2-adaptins (Fig. 2). The new human protein was named beta 3A-adaptin.


Fig. 1. Alignment of beta 3A-adaptin and beta -NAP amino acid sequences. The predicted amino acid sequences of human beta 3A-adaptin (GenBank accession code U81504[GenBank]) and human beta -NAP (GenBank accession code U37673[GenBank]) were aligned using the PILEUP program. Identical residues are boxed.
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Fig. 2. Comparison of the sequence of beta 3A-adaptin with those of other coat proteins. A, Kyte-Doolittle hydropathy plot of human beta 3A-adaptin. The sequence of beta 3A-adaptin was divided into three regions based on the hydropathy profile and the degree of identity to analogous regions in other adaptins. The three regions were designated A (amino-terminal), H (hinge), and C (carboxyl-terminal). B, schematic representation of the structure of human beta 3A-adaptin, beta -NAP, the S. cerevisiae (Sc) YGR261c gene product, and rat beta 1-adaptin, showing regions of sequence homology (corresponding domains are indicated by identical shading patterns). Amino acid numbers demarcating the different regions are shown on top of each scheme. C, a philogenetic tree comparing the sequence of human beta 3A-adaptin with those of other coat proteins from different species was constructed using the Darwin program (52). The length of each branch represents the philogenetic distance in PAM units (accepted point mutations per 100 residues); the bar represents 10 PAM units. The position of a putative ancestral precursor is indicated by the open circle. GenBank accession codes are: human beta -NAP, U37673[GenBank]; S. cerevisiae (Sc) YGR261c, Z73046[GenBank]; rat beta 1-adaptin, M77245[GenBank]; rat beta 2-adaptin, M34176[GenBank]; D. melanogaster (Dm) beta -adaptin, X75910[GenBank]; Sc ADB1, Z49505[GenBank]; Sc ADB2, Z28135[GenBank]; Sc beta -COP, Z49701[GenBank]; Dm beta -COP, L31852[GenBank]; rat beta -COP, X57228[GenBank].
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Based on a hydropathy plot, beta 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 beta 3A-adaptin to beta -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 beta -adaptins (Fig. 2B). In the beta 1- and beta 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 beta 1-adaptin, beta 2-adaptin, and beta -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 beta 3A-adaptin sequence reveals that this protein also contains 12 or 13 Arm repeats in the A region (not shown).

The H region of beta 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 beta 3A-adaptin is 35 and 19% identical to the analogous regions in beta -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 beta -adaptins was observed in this region, although the analogous segment in beta 1- and beta 2-adaptins is also hydrophilic and displays a hinge-like structure.

Finally, the C region of beta 3A-adaptin is 50% identical to the homologous segment of beta -NAP but shows no significant homology to beta 1- and beta 2-adaptins and is absent from YGR261c (Fig. 2B). In the beta -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, beta -COP, (Fig. 2C) shows that beta 3A-adaptin, beta -NAP, and YGR261c all belong to a group that may have diverged from the others early in the evolution of the family. The mammalian beta 1 and beta 2-adaptins, a D. melanogaster beta -adaptin and two other S. cerevisiae adaptins (Sc ADB1 and Sc ADB2) cluster together in a separate branch. The beta -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 (beta 3A-adaptin, beta -NAP, and YGR261c) that are more closely related to clathrin-associated beta -adaptins than to COPI components.

Tissue and Cell Expression of the beta 3A-adaptin mRNA

The pattern of expression of the beta 3A-adaptin mRNA in various human tissues and cell lines was examined by Northern blot analysis (Fig. 3). A single ~4.2-kb beta 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 beta 3A-adaptin mRNA, the beta -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 beta -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 beta -NAP mRNA in other neuronal cells but not in non-neuronal cells (Fig. 3C). Thus, the beta 3A-adaptin mRNA is widely expressed, whereas expression of the beta -NAP mRNA is restricted to brain and cell lines of neuronal origin.


Fig. 3. Analysis of beta 3A-adaptin mRNA expression in different tissues and cell lines. A, Northern blots with RNA from various human tissues were analyzed with a 32P-labeled probe specific for the beta 3A-adaptin mRNA. The positions of RNA size markers (in kb) are indicated at the left. A single transcript of ~4.2 kb was observed in all tissues. B, Northern blot analysis of the expression of the beta 3A-adaptin mRNA (top) and the beta -NAP mRNA (bottom) in various human cell lines. The sizes of the messages were ~4.2 and ~4.1 kb for beta 3A-adaptin and beta -NAP, respectively. C, analysis of the expression of the beta 3A-adaptin mRNA (top) and the beta -NAP mRNA (bottom) by RT-PCR. DNA was detected by staining with ethidium bromide.
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beta 3A-adaptin Is a Component of the AP-3 Complex

To characterize the biochemical properties of the beta 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 beta 3A-adaptin mRNA but not the beta -NAP mRNA (Fig. 3). Antibodies to both the H and C regions of beta 3A-adaptin (beta 3H7 and beta 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 sigma 3 (Fig. 4A), a known component of the AP-3 complex (30). The peak elution of beta 3A-adaptin and sigma 3 corresponded to a complex with Stokes radius of ~85 Å, as previously reported (30).


Fig. 4. The beta 3A-adaptin protein is a component of the AP-3 complex. A, cytosol from M1 cells was fractionated on a Superose 6 gel filtration column and fractions 28-36 were subjected to Western blot analysis with antibodies to sigma 3 and to the H (beta 3H7) and C (beta 3C1) regions of beta 3A-adaptin. Both beta 3A-adaptin and sigma 3 co-eluted in this column, peaking at a fraction corresponding to a Stokes radius of ~85 Å (30). B, the AP-3 complex was purified by affinity chromatography on an anti-sigma 3 antibody column. Western blots of the purified complex were probed with an irrelevant antibody (anti-FLAG), with an antibody to sigma 3, or with the anti-beta 3A-adaptin antibodies beta 3A1, beta 3H7, and beta 3C1. The positions of molecular markers (in kDa) are indicated at the left.
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To address directly the question of whether beta 3A-adaptin is a component of AP-3, this complex was affinity-purified from M1 cells on an anti-sigma 3 column and the eluate was analyzed by Western blotting with anti-sigma 3 and anti-beta 3A-adaptin antibodies (Fig. 4B). The anti-sigma 3 antibody recognized minor and major species of 20-22 kDa, as previously shown (30), whereas the different antibodies to beta 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-sigma 3 antibody (data not shown). Thus, beta 3A-adaptin co-purifies with sigma 3 on affinity chromatography.

The association of beta 3A-adaptin with sigma 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 sigma 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 beta 3H1 antibody (Fig. 5A) and with the antibodies beta 3H7, beta 3A1, and beta 3C1 (not shown) further confirmed that beta 3A-adaptin is indeed a component of the AP-3 complex. Fig. 5A also illustrates that, in addition to sigma 3 (A or B) and beta 3A-adaptin, the other components of AP-3 are p47A (27) and another protein known as delta -adaptin.2


Fig. 5. Alkaline phosphatase treatment affects the electrophoretic mobility of beta 3A-adaptin. A, the AP-3 complex was isolated from a cytosolic extract of [35S]methionine-labeled M1 cells by immunoprecipitation with an antibody to sigma 3. The complex was then dissociated in the presence of SDS and subjected to re-precipitation with antibodies to BSA (nonspecific control), delta -adaptin, beta 3A-adaptin, p47, and sigma 3, as indicated in the figure. The immunoprecipitates were treated for 1 h at 37 °C in the absence (-) or presence (+) of alkaline phosphatase and then resolved by SDS-PAGE on a 4-20% polyacrylamide gel. Notice the slight decrease in the electrophoretic mobility of beta 3A-adaptin upon treatment with alkaline phosphatase. Other subunits of AP-3 were not noticeably affected. B, analysis of the effect of alkaline phosphatase on the migration of [35S]methionine-labeled beta 3A-adaptin isolated from either cytosol or membranes. Samples were resolved by SDS-PAGE on a 8% acrylamide gel; this gel system allowed a better resolution of phosphorylated and dephosphorylated forms of beta 3A-adaptin. The positions of molecular markers (in kDa) are indicated on the left.
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beta 3A-adaptin Is a Phosphoprotein

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 beta 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 beta 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 beta 3A-adaptin is a phosphorylated protein at steady state. In contrast to beta 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 beta 3A-adaptin correlates with association to membranes. We found that both the cytosolic and membrane-bound forms of beta 3A-adaptin were equally sensitive to alkaline phosphatase (Fig. 5B), thus deeming it unlikely that the observed phosphorylation regulates membrane association.

To confirm that beta 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 beta 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 beta 3A-adaptin revealed that the phosphorylation was on serine residues (Fig. 6B). From these experiments, we concluded that beta 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.


Fig. 6. Labeling of beta 3A-adaptin with [32P]orthophosphate in vivo. A, M1 cells were metabolically labeled with [32P]orthophosphate and the AP-3 complex was isolated by a first (1st) immunoprecipitation with an antibody to sigma 3. A single 32P-labeled band of ~140 kDa was observed upon analysis of the immunoprecipitate by SDS-PAGE (lane 1). To identify this band and to examine the phosphorylation status of each of the AP-3 subunits, the labeled AP-3 complex was denatured in the presence of SDS and subjected to a second (2nd) immunoprecipitation with antibodies to BSA (lane 2), delta -adaptin (lane 3), beta 3A-adaptin (lane 4), p47 (lane 5), and sigma 3 (lane 6). The positions of molecular mass markers (in kDa) are indicated on the left. Notice that beta 3A-adaptin was the only AP-3 subunit that labeled with 32P under these conditions. B, 32P-labeled beta 3A-adaptin was isolated from a polyacrylamide gel such as that shown in A, and subjected to two-dimensional phosphoamino acid analysis as described under "Experimental Procedures." The migration of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) standards was visualized with ninhydrin. +, anode; -, cathode.
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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 beta 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 (beta 3H7) to the beta 3A-adaptin hinge that does not recognize beta -NAP. We performed a similar analysis using antibodies to two other components of the AP-3 complex, p47 (A and B) and sigma 3 (A and B), and to a component of AP-2, alpha -adaptin, as a control. Two forms of alpha -adaptin (alpha a and alpha 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 beta 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 sigma 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.


Fig. 7. AP-3 subunits are not enriched in clathrin-coated vesicles. A crude membrane fraction and purified clathrin-coated vesicles from bovine brain were analyzed for the presence of alpha -adaptin, beta 3A-adaptin, p47, and sigma 3 by Western blotting. The antibody to alpha -adaptin recognizes both the alpha a (~105 kDa) and alpha c (~102 kDa) forms of the protein (39). Similarly, the antibodies to sigma 3 and p47 react with both the A and B forms of these proteins (30). The antibody to beta 3A-adaptin (beta 3H7), on the other hand, is specific for this protein and does not cross-react with beta -NAP. Notice the enrichment of alpha -adaptin in the clathrin-coated vesicle fraction and the absence of p47, sigma 3, and beta 3A-adaptin from that fraction.
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DISCUSSION

In this study we describe a novel human protein named beta 3A-adaptin. The beta 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 beta 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 delta -adaptin,2 p47A (Ref. 27; now called µ3A), and sigma 3A or sigma 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 beta -NAP, which is also absent from clathrin-coated vesicles (28, 31).

The existence of a closely-related homolog of the brain-specific beta -NAP was first evidenced by the immunoprecipitation of a ~140-kDa protein from non-neuronal cells using an antibody to beta -NAP (30). Since beta -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 beta 3A-adaptin shares 61% overall identity and 75% overall similarity with beta -NAP. beta -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 beta 3A-adaptin-containing complex and the brain-specific beta -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 beta -NAP, other coat proteins display a lower but significant degree of homology to beta 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 beta 3A-adaptin and/or beta -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 beta 3A-adaptin also has a significant degree of homology to the clathrin-associated adaptor subunits beta 1- and beta 2-adaptins and to the COPI subunit beta -COP. The fact that beta 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 beta 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 beta 1- and beta 2-adaptins (40, 41). The A region is homologous to the head or core domains of beta 1- and beta 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 beta 1- and beta 2-adaptins, which mediate interactions with clathrin (7, 43). Finally, the C region of beta 3A-adaptin might be analogous to the ear or appendage domains of beta 1- and beta 2-adaptins. The function of the beta 1- and beta 2-adaptin ear domains is still unclear, although the analogous domain of alpha -adaptin has been shown to bind regulatory molecules such as dynamin (44) and Eps15 (45). The domain organization of delta -adaptin is also thought to resemble those of alpha - and gamma -adaptin.2 These similarities suggest that AP-3 may have an overall structure analogous to AP-1 and AP-2.

A salient feature of beta 3A-adaptin is that it is phosphorylated on serine residues (Figs. 5 and 6). Moreover, beta 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 beta 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 beta 3A-adaptin to beta -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, beta -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 beta 1-, beta 2-, and beta 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.


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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81504[GenBank].


Dagger    To whom correspondence should be addressed: CBMB, NICHD, National Institutes of Health, Bldg. 18T, Rm. 101, 18 Library Dr. MSC 5430, Bethesda, MD 20892-5430. Tel.: 301-496-6368; Fax: 301-402-0078; E-mail: juan{at}helix.nih.gov.
1   The abbreviations used are: RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; EST, expressed sequence tag; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT, reverse transcriptase; kb, kilobase pair(s).
2   C. E. Ooi, J. E. Moreira, E. C. Dell'Angelica, G. Poy, D. Wassarman, and J. S. Bonifacino, submitted for publication.
3   S. Lemmon and L. Robinson, personal communication.

ACKNOWLEDGEMENTS

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.


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