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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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Coimmunoprecipitation of AP-3 subunits. Pig brain cytosol was immunoprecipitated under nondenaturing conditions  with affinity-purified polyclonal antibodies against β3B, δ, σ3  (crossreacting with both the A and B isoforms), and γ. Gels were  blotted, and the appropriate region was cut out and probed with  each of the above antibodies, as well as with anti-μ3 (which does  not recognize the native complex). The four subunits of the AP-3  complex, δ, β3, μ3, and σ3, all coimmunoprecipitate; the γ subunit of the AP-1 complex does not coimmunoprecipitate with antibodies against the AP-3 components, and antibodies against γ  do not bring down any of these components. The doublet labeled  with anti-σ3 presumably corresponds to the A and B isoforms,  one of which appears to be preferentially immunoprecipitated  with this antibody.
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Figure 5: Coimmunoprecipitation of AP-3 subunits. Pig brain cytosol was immunoprecipitated under nondenaturing conditions with affinity-purified polyclonal antibodies against β3B, δ, σ3 (crossreacting with both the A and B isoforms), and γ. Gels were blotted, and the appropriate region was cut out and probed with each of the above antibodies, as well as with anti-μ3 (which does not recognize the native complex). The four subunits of the AP-3 complex, δ, β3, μ3, and σ3, all coimmunoprecipitate; the γ subunit of the AP-1 complex does not coimmunoprecipitate with antibodies against the AP-3 components, and antibodies against γ do not bring down any of these components. The doublet labeled with anti-σ3 presumably corresponds to the A and B isoforms, one of which appears to be preferentially immunoprecipitated with this antibody.

Mentions: To characterize δ and σ3 further and to find out whether they are in fact associated with μ3 and β3, portions of them were expressed as fusion proteins and used to raise antibodies in rabbits. The antibodies were then used in immunoprecipitation and Western blotting experiments, together with the previously described antibodies against μ3 and β3. Fig. 5 shows the results of one such experiment. Pig brain cytosol was immunoprecipitated under nondenaturing conditions with anti-β3, anti-δ, and anti-σ3 (using pooled antibodies raised against both the A and B isoforms). As a control, cytosol was also immunoprecipitated with anti–γ-adaptin to bring down the AP-1 adaptor complex. Strips were then cut from Western blots and probed with antibodies against δ, β3, μ3, σ3, and γ.


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

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

Coimmunoprecipitation of AP-3 subunits. Pig brain cytosol was immunoprecipitated under nondenaturing conditions  with affinity-purified polyclonal antibodies against β3B, δ, σ3  (crossreacting with both the A and B isoforms), and γ. Gels were  blotted, and the appropriate region was cut out and probed with  each of the above antibodies, as well as with anti-μ3 (which does  not recognize the native complex). The four subunits of the AP-3  complex, δ, β3, μ3, and σ3, all coimmunoprecipitate; the γ subunit of the AP-1 complex does not coimmunoprecipitate with antibodies against the AP-3 components, and antibodies against γ  do not bring down any of these components. The doublet labeled  with anti-σ3 presumably corresponds to the A and B isoforms,  one of which appears to be preferentially immunoprecipitated  with this antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139840&req=5

Figure 5: Coimmunoprecipitation of AP-3 subunits. Pig brain cytosol was immunoprecipitated under nondenaturing conditions with affinity-purified polyclonal antibodies against β3B, δ, σ3 (crossreacting with both the A and B isoforms), and γ. Gels were blotted, and the appropriate region was cut out and probed with each of the above antibodies, as well as with anti-μ3 (which does not recognize the native complex). The four subunits of the AP-3 complex, δ, β3, μ3, and σ3, all coimmunoprecipitate; the γ subunit of the AP-1 complex does not coimmunoprecipitate with antibodies against the AP-3 components, and antibodies against γ do not bring down any of these components. The doublet labeled with anti-σ3 presumably corresponds to the A and B isoforms, one of which appears to be preferentially immunoprecipitated with this antibody.
Mentions: To characterize δ and σ3 further and to find out whether they are in fact associated with μ3 and β3, portions of them were expressed as fusion proteins and used to raise antibodies in rabbits. The antibodies were then used in immunoprecipitation and Western blotting experiments, together with the previously described antibodies against μ3 and β3. Fig. 5 shows the results of one such experiment. Pig brain cytosol was immunoprecipitated under nondenaturing conditions with anti-β3, anti-δ, and anti-σ3 (using pooled antibodies raised against both the A and B isoforms). As a control, cytosol was also immunoprecipitated with anti–γ-adaptin to bring down the AP-1 adaptor complex. Strips were then cut from Western blots and probed with antibodies against δ, β3, μ3, σ3, and γ.

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