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Assembly and function of AP-3 complexes in cells expressing mutant subunits.

Peden AA, Rudge RE, Lui WW, Robinson MS - J. Cell Biol. (2002)

Bottom Line: The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other.However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting.These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

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Immunoprecipitates and Western blots of AP-3 subunits from mh and pe cells. Extracts of cultured mh cells (left), cultured pe cells (middle), and spleens from both pe and control mice (right) were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions, with the exception of the anti-μ3 immunoprecipitations, which were performed in the presence of SDS. The gels were then blotted onto nitrocellulose and probed with the indicated antibody. An antibody against the AP-1 γ subunit was included as a control. The mh cells can be seen to express trace amounts of β3 and μ3, which form a dimer, whereas the σ3 subunit appears to be monomeric. The δ and σ3 expressed in the pe cells also form a dimer, and in addition a small amount of complete heterotetramer is made. The various subcomplexes that form in the mh and pe cells are indicated diagrammatically.
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fig2: Immunoprecipitates and Western blots of AP-3 subunits from mh and pe cells. Extracts of cultured mh cells (left), cultured pe cells (middle), and spleens from both pe and control mice (right) were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions, with the exception of the anti-μ3 immunoprecipitations, which were performed in the presence of SDS. The gels were then blotted onto nitrocellulose and probed with the indicated antibody. An antibody against the AP-1 γ subunit was included as a control. The mh cells can be seen to express trace amounts of β3 and μ3, which form a dimer, whereas the σ3 subunit appears to be monomeric. The δ and σ3 expressed in the pe cells also form a dimer, and in addition a small amount of complete heterotetramer is made. The various subcomplexes that form in the mh and pe cells are indicated diagrammatically.

Mentions: Fig. 2 (left) shows that under these conditions both β3 and μ3 can be detected in the mh cells, and furthermore that they assemble into a heterodimer since both subunits come down with anti-β3. In contrast, the σ3 in these cells appears to exist as a monomer, since it does not coimmunoprecipitate with any of the other subunits. In the pe cells (Fig. 2, middle), δ and σ3 form heterodimers which come down with antibodies against both subunits, and in addition, a small amount of μ3 can be detected which coprecipitates with the δ/σ3 dimers. This suggests either that the μ3 may be assembling with the δ and σ3 subunits into a heterotrimer, or alternatively, since the pe mutation is not a true , that there are small amounts of truncated β3A expressed in these cells which can form heterotetramers. To distinguish between these two possibilities, we immunoprecipitated extracts from pe spleen under nondenaturing conditions with a β3A-specific antibody (the reason for using spleen for this experiment is that it is possible to get a more highly concentrated extract from tissues than from cultured cells, and AP-3 is known to be expressed at particularly high levels in spleen). Fig. 2 (right) demonstrates that there are indeed small amounts of truncated β3A made in pe cells which can coassemble with the other three subunits to form a heterotetramer, although we cannot rule out the possibility that some of the μ3 may also be assembled into a heterotrimer. Together, these studies indicate that β3 and μ3 can interact with each other, forming heterodimers in mh cells, and that δ and σ3 also interact with each other, forming heterodimers in pe cells. The interactions that we can detect in the mutant cells are shown diagrammatically in the bottom part of Fig. 2.


Assembly and function of AP-3 complexes in cells expressing mutant subunits.

Peden AA, Rudge RE, Lui WW, Robinson MS - J. Cell Biol. (2002)

Immunoprecipitates and Western blots of AP-3 subunits from mh and pe cells. Extracts of cultured mh cells (left), cultured pe cells (middle), and spleens from both pe and control mice (right) were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions, with the exception of the anti-μ3 immunoprecipitations, which were performed in the presence of SDS. The gels were then blotted onto nitrocellulose and probed with the indicated antibody. An antibody against the AP-1 γ subunit was included as a control. The mh cells can be seen to express trace amounts of β3 and μ3, which form a dimer, whereas the σ3 subunit appears to be monomeric. The δ and σ3 expressed in the pe cells also form a dimer, and in addition a small amount of complete heterotetramer is made. The various subcomplexes that form in the mh and pe cells are indicated diagrammatically.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199225&req=5

fig2: Immunoprecipitates and Western blots of AP-3 subunits from mh and pe cells. Extracts of cultured mh cells (left), cultured pe cells (middle), and spleens from both pe and control mice (right) were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions, with the exception of the anti-μ3 immunoprecipitations, which were performed in the presence of SDS. The gels were then blotted onto nitrocellulose and probed with the indicated antibody. An antibody against the AP-1 γ subunit was included as a control. The mh cells can be seen to express trace amounts of β3 and μ3, which form a dimer, whereas the σ3 subunit appears to be monomeric. The δ and σ3 expressed in the pe cells also form a dimer, and in addition a small amount of complete heterotetramer is made. The various subcomplexes that form in the mh and pe cells are indicated diagrammatically.
Mentions: Fig. 2 (left) shows that under these conditions both β3 and μ3 can be detected in the mh cells, and furthermore that they assemble into a heterodimer since both subunits come down with anti-β3. In contrast, the σ3 in these cells appears to exist as a monomer, since it does not coimmunoprecipitate with any of the other subunits. In the pe cells (Fig. 2, middle), δ and σ3 form heterodimers which come down with antibodies against both subunits, and in addition, a small amount of μ3 can be detected which coprecipitates with the δ/σ3 dimers. This suggests either that the μ3 may be assembling with the δ and σ3 subunits into a heterotrimer, or alternatively, since the pe mutation is not a true , that there are small amounts of truncated β3A expressed in these cells which can form heterotetramers. To distinguish between these two possibilities, we immunoprecipitated extracts from pe spleen under nondenaturing conditions with a β3A-specific antibody (the reason for using spleen for this experiment is that it is possible to get a more highly concentrated extract from tissues than from cultured cells, and AP-3 is known to be expressed at particularly high levels in spleen). Fig. 2 (right) demonstrates that there are indeed small amounts of truncated β3A made in pe cells which can coassemble with the other three subunits to form a heterotetramer, although we cannot rule out the possibility that some of the μ3 may also be assembled into a heterotrimer. Together, these studies indicate that β3 and μ3 can interact with each other, forming heterodimers in mh cells, and that δ and σ3 also interact with each other, forming heterodimers in pe cells. The interactions that we can detect in the mutant cells are shown diagrammatically in the bottom part of Fig. 2.

Bottom Line: The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other.However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting.These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

Show MeSH
Related in: MedlinePlus