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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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Expression patterns of δ, β3A, σ3A, and σ3B. Northern  blots were probed with either oligonucleotides or cDNAs specific  for each of the four sequences. All four genes are expressed ubiquitously. The relative weakness of the signal obtained with β3A  probe is probably a consequence of the blot already having been  probed several times.
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Figure 4: Expression patterns of δ, β3A, σ3A, and σ3B. Northern blots were probed with either oligonucleotides or cDNAs specific for each of the four sequences. All four genes are expressed ubiquitously. The relative weakness of the signal obtained with β3A probe is probably a consequence of the blot already having been probed several times.

Mentions: Both μ3 and β3 have neuronal-specific isoforms, so we probed multiple tissue Northern blots to find out whether any of the novel sequences also show tissue-specific expression. Fig. 4 shows that δ, β3A, σ3A, and σ3B are all expressed ubiquitously. Thus, β3A is indeed a nonneuronal-specific isoform of β3; the only known isoform of δ is expressed in all tissues examined; and although there are two isoforms of σ3, they have similar expression patterns.


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

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

Expression patterns of δ, β3A, σ3A, and σ3B. Northern  blots were probed with either oligonucleotides or cDNAs specific  for each of the four sequences. All four genes are expressed ubiquitously. The relative weakness of the signal obtained with β3A  probe is probably a consequence of the blot already having been  probed several times.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Expression patterns of δ, β3A, σ3A, and σ3B. Northern blots were probed with either oligonucleotides or cDNAs specific for each of the four sequences. All four genes are expressed ubiquitously. The relative weakness of the signal obtained with β3A probe is probably a consequence of the blot already having been probed several times.
Mentions: Both μ3 and β3 have neuronal-specific isoforms, so we probed multiple tissue Northern blots to find out whether any of the novel sequences also show tissue-specific expression. Fig. 4 shows that δ, β3A, σ3A, and σ3B are all expressed ubiquitously. Thus, β3A is indeed a nonneuronal-specific isoform of β3; the only known isoform of δ is expressed in all tissues examined; and although there are two isoforms of σ3, they have similar expression patterns.

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