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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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AP-3 subunits in mh and pe cells. (a) Western blots of lysates from the mh and pe cells, as well as a wild-type control cell line (melan-a cells), were probed with antibodies against all four AP-3 subunits and with an antibody against the AP-1 γ subunit as a control. Only the σ3 subunit is detectable in the mh cells under these conditions, while only the δ and σ3 subunits are detectable in the pe cells. (b–d) The same cells were labeled for immunofluorescence with an antibody against the AP-3 δ subunit. Control cells (b) have the typical punctate pattern, mh cells (c) show no labeling, and pe cells (d) have diffuse cytoplasmic labeling, indicating that although the δ subunit is detectable, it is cytosolic rather than membrane-associated. Bar, 20 μm.
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fig1: AP-3 subunits in mh and pe cells. (a) Western blots of lysates from the mh and pe cells, as well as a wild-type control cell line (melan-a cells), were probed with antibodies against all four AP-3 subunits and with an antibody against the AP-1 γ subunit as a control. Only the σ3 subunit is detectable in the mh cells under these conditions, while only the δ and σ3 subunits are detectable in the pe cells. (b–d) The same cells were labeled for immunofluorescence with an antibody against the AP-3 δ subunit. Control cells (b) have the typical punctate pattern, mh cells (c) show no labeling, and pe cells (d) have diffuse cytoplasmic labeling, indicating that although the δ subunit is detectable, it is cytosolic rather than membrane-associated. Bar, 20 μm.

Mentions: To investigate the mocha (mh) and pearl (pe) phenotypes at the cellular level, we have established an mh cell line and made use of a previously established pe cell line. Fig. 1 a shows Western blots of extracts from these cells probed for the four AP-3 subunits: δ, β3, μ3, and σ3. In the cells from the mocha homozygote (mh/mh), δ, β3, and μ3 are all undetectable, while σ3 is detectable but at reduced levels. In the pearl cells (pe/pe), β3 and μ3 are both undetectable, while δ and σ3 are both detectable at reduced levels. When the cells are labeled for immunofluorescence using a δ-specific antibody, the typical punctate pattern can be seen in control cells (+), the mh cells are unlabeled, and the pe cells show diffuse cytoplasmic labeling, indicating that although δ is present in pe cells, it is unable to associate with membranes in the absence of the β3 and/or μ3 subunits (Fig. 1, b–d).


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

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

AP-3 subunits in mh and pe cells. (a) Western blots of lysates from the mh and pe cells, as well as a wild-type control cell line (melan-a cells), were probed with antibodies against all four AP-3 subunits and with an antibody against the AP-1 γ subunit as a control. Only the σ3 subunit is detectable in the mh cells under these conditions, while only the δ and σ3 subunits are detectable in the pe cells. (b–d) The same cells were labeled for immunofluorescence with an antibody against the AP-3 δ subunit. Control cells (b) have the typical punctate pattern, mh cells (c) show no labeling, and pe cells (d) have diffuse cytoplasmic labeling, indicating that although the δ subunit is detectable, it is cytosolic rather than membrane-associated. Bar, 20 μm.
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Related In: Results  -  Collection

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fig1: AP-3 subunits in mh and pe cells. (a) Western blots of lysates from the mh and pe cells, as well as a wild-type control cell line (melan-a cells), were probed with antibodies against all four AP-3 subunits and with an antibody against the AP-1 γ subunit as a control. Only the σ3 subunit is detectable in the mh cells under these conditions, while only the δ and σ3 subunits are detectable in the pe cells. (b–d) The same cells were labeled for immunofluorescence with an antibody against the AP-3 δ subunit. Control cells (b) have the typical punctate pattern, mh cells (c) show no labeling, and pe cells (d) have diffuse cytoplasmic labeling, indicating that although the δ subunit is detectable, it is cytosolic rather than membrane-associated. Bar, 20 μm.
Mentions: To investigate the mocha (mh) and pearl (pe) phenotypes at the cellular level, we have established an mh cell line and made use of a previously established pe cell line. Fig. 1 a shows Western blots of extracts from these cells probed for the four AP-3 subunits: δ, β3, μ3, and σ3. In the cells from the mocha homozygote (mh/mh), δ, β3, and μ3 are all undetectable, while σ3 is detectable but at reduced levels. In the pearl cells (pe/pe), β3 and μ3 are both undetectable, while δ and σ3 are both detectable at reduced levels. When the cells are labeled for immunofluorescence using a δ-specific antibody, the typical punctate pattern can be seen in control cells (+), the mh cells are unlabeled, and the pe cells show diffuse cytoplasmic labeling, indicating that although δ is present in pe cells, it is unable to associate with membranes in the absence of the β3 and/or μ3 subunits (Fig. 1, b–d).

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