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Characterization of the adaptor-related protein complex, AP-3.

Simpson F, Peden AA, Christopoulou L, Robinson MS - J. Cell Biol. (1997)

Bottom Line: Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures.The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues.Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge CB2 2QR, United Kingdom.

ABSTRACT
We have recently shown that two proteins related to two of the adaptor subunits of clathrincoated vesicles, p47 (mu3) and beta-NAP (beta3B), are part of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell Biol. 133:749-760). In the present study we have searched the EST database and have identified, cloned, and sequenced a ubiquitously expressed homologue of beta-NAP, beta3A, as well as homologues of the alpha/gamma and sigma adaptor subunits, delta and sigma3, which are also ubiquitously expressed. Antibodies raised against recombinant delta and sigma3 show that they are the other two subunits of the adaptor-like complex. We are calling this complex AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a more appropriate designation for an adaptor-related heterotetramer. Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only limited colocalization with endosomal markers and may correspond to a postTGN biosynthetic compartment. The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues. Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

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Diagrams of the two conventional adaptor complexes,  together with the adaptor-related complex, AP-3. The AP-1 complex is associated with the TGN, the AP-2 complex is associated  with the plasma membrane, and the AP-3 complex also appears  to be associated with the TGN as well as with more peripheral  membranes. Each complex consists of four subunits, belonging to  four different families. γ, α, and δ are related; β1 (β′), β2 (β), and  β3 (β-NAP/β3B and β3A) are related; μ1 (AP47), μ2 (AP50),  and μ3 (p47A/μ3A and p47B/μ3B) are related; and σ1 (AP19),  σ2 (AP17), and σ3 (A and B) are related. EM studies of the AP-2  complex have revealed that it has a structure resembling a head  flanked by two ears connected by flexible hinges (Heuser and  Keen, 1988), and although such studies have not yet been carried  out on AP-1 or AP-3, the sequence homologies suggest that all  three complexes have a similar structure.
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Figure 1: Diagrams of the two conventional adaptor complexes, together with the adaptor-related complex, AP-3. The AP-1 complex is associated with the TGN, the AP-2 complex is associated with the plasma membrane, and the AP-3 complex also appears to be associated with the TGN as well as with more peripheral membranes. Each complex consists of four subunits, belonging to four different families. γ, α, and δ are related; β1 (β′), β2 (β), and β3 (β-NAP/β3B and β3A) are related; μ1 (AP47), μ2 (AP50), and μ3 (p47A/μ3A and p47B/μ3B) are related; and σ1 (AP19), σ2 (AP17), and σ3 (A and B) are related. EM studies of the AP-2 complex have revealed that it has a structure resembling a head flanked by two ears connected by flexible hinges (Heuser and Keen, 1988), and although such studies have not yet been carried out on AP-1 or AP-3, the sequence homologies suggest that all three complexes have a similar structure.

Mentions: Immunoprecipitation experiments indicated that the complex consists not only of β-NAP and p47, but also of two other proteins of ∼160 (p160) and ∼25 kD (p25), making it a heterotetramer like an adaptor complex. A model for this complex is shown in Fig. 1, together with models of the AP-1 and AP-2 complexes. We are calling this complex AP-3, by analogy with AP-1 and AP-2. The name AP-3 has also been used for AP180, a clathrin assembly–promoting phosphoprotein specifically expressed in neurons (Keen, 1987; Morris et al., 1993). However, AP180 is a monomer that does not show any sequence homology with the adaptor subunits, so it seems more appropriate to use this name for an adaptor-related heterotetrameric complex. Both AP-1 and AP-2 consist of four subunits: γ- or α-adaptin; β1- or β2-adaptin (also called β′ and β); μ1 (AP47) or μ2 (AP50); and σ1 (AP19) or σ2 (AP17). The two subunits of AP-3 that have already been identified, p47 and β-NAP, are labeled μ3 and β3 on the diagram. The other two proteins that coimmunoprecipitate with μ3 and β3, p160 and p25, are labeled δ and σ3, respectively. We predicted that δ would turn out to be a homologue of the α- and γ-adaptins and that σ3 would turn out to be a homologue of σ1 and σ2 (Simpson et al., 1996). In the EST database, there are candidates for both of these proteins as well as a candidate for a ubiquitously expressed isoform of β-NAP. We have now raised antibodies against the δ and σ3 candidates expressed as fusion proteins, and here we show by coimmunoprecipitation that they are indeed the missing subunits of the complex. To learn more about the function of the AP-3 complex we have used the antibodies to localize endogenous AP-3 in nonneuronal cells and to compare its distribution with that of other proteins. In addition, we have found a close homologue of the δ subunit in Drosophila, which is encoded by the garnet gene. Studying the mutant phenotype of the gene provides additional insights into the role of the complex.


Characterization of the adaptor-related protein complex, AP-3.

Simpson F, Peden AA, Christopoulou L, Robinson MS - J. Cell Biol. (1997)

Diagrams of the two conventional adaptor complexes,  together with the adaptor-related complex, AP-3. The AP-1 complex is associated with the TGN, the AP-2 complex is associated  with the plasma membrane, and the AP-3 complex also appears  to be associated with the TGN as well as with more peripheral  membranes. Each complex consists of four subunits, belonging to  four different families. γ, α, and δ are related; β1 (β′), β2 (β), and  β3 (β-NAP/β3B and β3A) are related; μ1 (AP47), μ2 (AP50),  and μ3 (p47A/μ3A and p47B/μ3B) are related; and σ1 (AP19),  σ2 (AP17), and σ3 (A and B) are related. EM studies of the AP-2  complex have revealed that it has a structure resembling a head  flanked by two ears connected by flexible hinges (Heuser and  Keen, 1988), and although such studies have not yet been carried  out on AP-1 or AP-3, the sequence homologies suggest that all  three complexes have a similar structure.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139840&req=5

Figure 1: Diagrams of the two conventional adaptor complexes, together with the adaptor-related complex, AP-3. The AP-1 complex is associated with the TGN, the AP-2 complex is associated with the plasma membrane, and the AP-3 complex also appears to be associated with the TGN as well as with more peripheral membranes. Each complex consists of four subunits, belonging to four different families. γ, α, and δ are related; β1 (β′), β2 (β), and β3 (β-NAP/β3B and β3A) are related; μ1 (AP47), μ2 (AP50), and μ3 (p47A/μ3A and p47B/μ3B) are related; and σ1 (AP19), σ2 (AP17), and σ3 (A and B) are related. EM studies of the AP-2 complex have revealed that it has a structure resembling a head flanked by two ears connected by flexible hinges (Heuser and Keen, 1988), and although such studies have not yet been carried out on AP-1 or AP-3, the sequence homologies suggest that all three complexes have a similar structure.
Mentions: Immunoprecipitation experiments indicated that the complex consists not only of β-NAP and p47, but also of two other proteins of ∼160 (p160) and ∼25 kD (p25), making it a heterotetramer like an adaptor complex. A model for this complex is shown in Fig. 1, together with models of the AP-1 and AP-2 complexes. We are calling this complex AP-3, by analogy with AP-1 and AP-2. The name AP-3 has also been used for AP180, a clathrin assembly–promoting phosphoprotein specifically expressed in neurons (Keen, 1987; Morris et al., 1993). However, AP180 is a monomer that does not show any sequence homology with the adaptor subunits, so it seems more appropriate to use this name for an adaptor-related heterotetrameric complex. Both AP-1 and AP-2 consist of four subunits: γ- or α-adaptin; β1- or β2-adaptin (also called β′ and β); μ1 (AP47) or μ2 (AP50); and σ1 (AP19) or σ2 (AP17). The two subunits of AP-3 that have already been identified, p47 and β-NAP, are labeled μ3 and β3 on the diagram. The other two proteins that coimmunoprecipitate with μ3 and β3, p160 and p25, are labeled δ and σ3, respectively. We predicted that δ would turn out to be a homologue of the α- and γ-adaptins and that σ3 would turn out to be a homologue of σ1 and σ2 (Simpson et al., 1996). In the EST database, there are candidates for both of these proteins as well as a candidate for a ubiquitously expressed isoform of β-NAP. We have now raised antibodies against the δ and σ3 candidates expressed as fusion proteins, and here we show by coimmunoprecipitation that they are indeed the missing subunits of the complex. To learn more about the function of the AP-3 complex we have used the antibodies to localize endogenous AP-3 in nonneuronal cells and to compare its distribution with that of other proteins. In addition, we have found a close homologue of the δ subunit in Drosophila, which is encoded by the garnet gene. Studying the mutant phenotype of the gene provides additional insights into the role of the complex.

Bottom Line: Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures.The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues.Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge CB2 2QR, United Kingdom.

ABSTRACT
We have recently shown that two proteins related to two of the adaptor subunits of clathrincoated vesicles, p47 (mu3) and beta-NAP (beta3B), are part of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell Biol. 133:749-760). In the present study we have searched the EST database and have identified, cloned, and sequenced a ubiquitously expressed homologue of beta-NAP, beta3A, as well as homologues of the alpha/gamma and sigma adaptor subunits, delta and sigma3, which are also ubiquitously expressed. Antibodies raised against recombinant delta and sigma3 show that they are the other two subunits of the adaptor-like complex. We are calling this complex AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a more appropriate designation for an adaptor-related heterotetramer. Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only limited colocalization with endosomal markers and may correspond to a postTGN biosynthetic compartment. The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues. Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

Show MeSH
Related in: MedlinePlus