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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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Diagon plots comparing related proteins in the AP-1,  AP-2, and AP-3 complexes. The sequences of all four types of  subunits were compared using the “SIP” program (Staden, 1990),  which was also used to calculate the percent identity. The δ subunit shows the least similarity with its counterparts in the AP-1  and AP-2 complexes, while the σ subunits (σ3A and σ3B) show  the most. The protein product of the Drosophila garnet gene is  also shown, compared with the δ subunit.
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Figure 3: Diagon plots comparing related proteins in the AP-1, AP-2, and AP-3 complexes. The sequences of all four types of subunits were compared using the “SIP” program (Staden, 1990), which was also used to calculate the percent identity. The δ subunit shows the least similarity with its counterparts in the AP-1 and AP-2 complexes, while the σ subunits (σ3A and σ3B) show the most. The protein product of the Drosophila garnet gene is also shown, compared with the δ subunit.

Mentions: To find the missing subunits of the AP-3 complex we searched the EST database for likely candidates. Both μ3 and β3 are more distantly related to μ1/μ2 and β1/β2 than μ1 and μ2 or β1 and β2 are to each other (Fig. 1 and see Fig. 3); thus, we looked for proteins with a similar type of relationship to α- and γ-adaptin and to σ1 and σ2, as candidates for the δ and σ3 subunits. We also looked for a nonneuronal-specific homologue of β-NAP. cDNAs with the appropriate properties were then obtained and sequenced. In some cases the cDNAs were not full length, and it was necessary to carry out library screens to obtain the complete coding sequence.


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

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

Diagon plots comparing related proteins in the AP-1,  AP-2, and AP-3 complexes. The sequences of all four types of  subunits were compared using the “SIP” program (Staden, 1990),  which was also used to calculate the percent identity. The δ subunit shows the least similarity with its counterparts in the AP-1  and AP-2 complexes, while the σ subunits (σ3A and σ3B) show  the most. The protein product of the Drosophila garnet gene is  also shown, compared with the δ subunit.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139840&req=5

Figure 3: Diagon plots comparing related proteins in the AP-1, AP-2, and AP-3 complexes. The sequences of all four types of subunits were compared using the “SIP” program (Staden, 1990), which was also used to calculate the percent identity. The δ subunit shows the least similarity with its counterparts in the AP-1 and AP-2 complexes, while the σ subunits (σ3A and σ3B) show the most. The protein product of the Drosophila garnet gene is also shown, compared with the δ subunit.
Mentions: To find the missing subunits of the AP-3 complex we searched the EST database for likely candidates. Both μ3 and β3 are more distantly related to μ1/μ2 and β1/β2 than μ1 and μ2 or β1 and β2 are to each other (Fig. 1 and see Fig. 3); thus, we looked for proteins with a similar type of relationship to α- and γ-adaptin and to σ1 and σ2, as candidates for the δ and σ3 subunits. We also looked for a nonneuronal-specific homologue of β-NAP. cDNAs with the appropriate properties were then obtained and sequenced. In some cases the cDNAs were not full length, and it was necessary to carry out library screens to obtain the complete coding sequence.

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