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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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Confocal micrographs showing double labeling for AP complexes and clathrin. pe cells expressing β3A (a), β3Aβ2 (c), or β3A817AAA were double labeled with anti-δ (green) and anti-clathrin (red), and COS cells were double labeled with anti-γ (green) and anti-clathrin (red). Essentially all of the anti-γ labeling is also positive for clathrin (note how there are no green dots in b, only yellow). In contrast, in all three cell lines expressing β3 constructs, much of the AP-3 labeling is negative for clathrin. (Transfected pe cells could not be double labeled with anti-γ and anti-clathrin because the anti-γ mAb only recognizes the protein in nonrodent cells.) Bar: (a and b) 10 μm; (c and d) 20 μm.
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fig7: Confocal micrographs showing double labeling for AP complexes and clathrin. pe cells expressing β3A (a), β3Aβ2 (c), or β3A817AAA were double labeled with anti-δ (green) and anti-clathrin (red), and COS cells were double labeled with anti-γ (green) and anti-clathrin (red). Essentially all of the anti-γ labeling is also positive for clathrin (note how there are no green dots in b, only yellow). In contrast, in all three cell lines expressing β3 constructs, much of the AP-3 labeling is negative for clathrin. (Transfected pe cells could not be double labeled with anti-γ and anti-clathrin because the anti-γ mAb only recognizes the protein in nonrodent cells.) Bar: (a and b) 10 μm; (c and d) 20 μm.

Mentions: To determine whether the complexes could be recruited onto membranes, cells expressing each of the six constructs were labeled for immunofluorescence with anti-δ. In nontransfected pe cells, δ has a diffuse cytoplasmic distribution (Fig. 1 d); however, in cells expressing each of the five constructs, the δ labeling was punctate, indicating that it was associated with membranes (Fig. 6) . We also compared the distribution of AP-3 in cells expressing the various constructs with that of clathrin, using a confocal microscope (Fig. 7 ; AP-3 is green and clathrin is red). In cells expressing wild-type β3A, AP-3 and clathrin were found to have distinct distributions: (a) they are often in the same general vicinity, and in some cases there may be overlap (yellow), but much of the AP-3 labeling is negative for clathrin. In contrast, when cells are double labeled for the AP-1 adaptor complex and clathrin (b), essentially all of the structures that are positive for AP-1 (green) are also positive for clathrin (red; note that there is extensive yellow labeling and very little green). In cells expressing the various constructs, the extent of overlap between AP-3 and clathrin was very similar to that in cells expressing wild-type β3A. This is in spite of the fact that the β3Aβ2 chimera (c) has more potential clathrin binding sites than wild-type β3A (not only the clathrin-binding consensus sequence LLNLD in its hinge domain, but also three copies of another motif implicated in clathrin binding, DLL [Morgan et al., 2000], as well as clathrin binding activity in its ear domain [Owen et al., 2000]). Constructs lacking a clathrin binding domain, like β3A817AAA (d), did not look appreciably different from the other constructs: again, there were some yellow structures, as well as red and green. However, at the light microscope level, it is impossible to resolve two structures that are less than ∼0.2 μm apart from each other, so to quantify the true extent of overlap between AP-3 and clathrin in cells expressing the various constructs, it will be necessary to go to the electron microscope level.


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

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

Confocal micrographs showing double labeling for AP complexes and clathrin. pe cells expressing β3A (a), β3Aβ2 (c), or β3A817AAA were double labeled with anti-δ (green) and anti-clathrin (red), and COS cells were double labeled with anti-γ (green) and anti-clathrin (red). Essentially all of the anti-γ labeling is also positive for clathrin (note how there are no green dots in b, only yellow). In contrast, in all three cell lines expressing β3 constructs, much of the AP-3 labeling is negative for clathrin. (Transfected pe cells could not be double labeled with anti-γ and anti-clathrin because the anti-γ mAb only recognizes the protein in nonrodent cells.) Bar: (a and b) 10 μm; (c and d) 20 μm.
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fig7: Confocal micrographs showing double labeling for AP complexes and clathrin. pe cells expressing β3A (a), β3Aβ2 (c), or β3A817AAA were double labeled with anti-δ (green) and anti-clathrin (red), and COS cells were double labeled with anti-γ (green) and anti-clathrin (red). Essentially all of the anti-γ labeling is also positive for clathrin (note how there are no green dots in b, only yellow). In contrast, in all three cell lines expressing β3 constructs, much of the AP-3 labeling is negative for clathrin. (Transfected pe cells could not be double labeled with anti-γ and anti-clathrin because the anti-γ mAb only recognizes the protein in nonrodent cells.) Bar: (a and b) 10 μm; (c and d) 20 μm.
Mentions: To determine whether the complexes could be recruited onto membranes, cells expressing each of the six constructs were labeled for immunofluorescence with anti-δ. In nontransfected pe cells, δ has a diffuse cytoplasmic distribution (Fig. 1 d); however, in cells expressing each of the five constructs, the δ labeling was punctate, indicating that it was associated with membranes (Fig. 6) . We also compared the distribution of AP-3 in cells expressing the various constructs with that of clathrin, using a confocal microscope (Fig. 7 ; AP-3 is green and clathrin is red). In cells expressing wild-type β3A, AP-3 and clathrin were found to have distinct distributions: (a) they are often in the same general vicinity, and in some cases there may be overlap (yellow), but much of the AP-3 labeling is negative for clathrin. In contrast, when cells are double labeled for the AP-1 adaptor complex and clathrin (b), essentially all of the structures that are positive for AP-1 (green) are also positive for clathrin (red; note that there is extensive yellow labeling and very little green). In cells expressing the various constructs, the extent of overlap between AP-3 and clathrin was very similar to that in cells expressing wild-type β3A. This is in spite of the fact that the β3Aβ2 chimera (c) has more potential clathrin binding sites than wild-type β3A (not only the clathrin-binding consensus sequence LLNLD in its hinge domain, but also three copies of another motif implicated in clathrin binding, DLL [Morgan et al., 2000], as well as clathrin binding activity in its ear domain [Owen et al., 2000]). Constructs lacking a clathrin binding domain, like β3A817AAA (d), did not look appreciably different from the other constructs: again, there were some yellow structures, as well as red and green. However, at the light microscope level, it is impossible to resolve two structures that are less than ∼0.2 μm apart from each other, so to quantify the true extent of overlap between AP-3 and clathrin in cells expressing the various constructs, it will be necessary to go to the electron microscope level.

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