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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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Related in: MedlinePlus

Subunit interactions between AP-3 subunits detected using the yeast two-hybrid system. Yeast cells were transformed with cDNAs in either pGBT9 or pGAD424, as indicated, and interactions were assayed by growth in the absence of histidine. Panels a and b show that the δ subunit interacts with both σ3A and σ3B, panel c shows that the β3A subunit interacts with both μ3A and μ3B, and panel d shows that the β3B subunit interacts with μ3A.
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fig3: Subunit interactions between AP-3 subunits detected using the yeast two-hybrid system. Yeast cells were transformed with cDNAs in either pGBT9 or pGAD424, as indicated, and interactions were assayed by growth in the absence of histidine. Panels a and b show that the δ subunit interacts with both σ3A and σ3B, panel c shows that the β3A subunit interacts with both μ3A and μ3B, and panel d shows that the β3B subunit interacts with μ3A.

Mentions: To investigate interactions between the AP-3 subunits further, and also to determine whether the A and B isoforms of the various subunits can associate with each other, we cloned the cDNAs into the vectors pGBT9 and pGAD424 for analysis using the yeast two-hybrid system. Fig. 3 , a and b, shows that δ interacts under these conditions with σ3A and σ3B, both of which are expressed ubiquitously. In addition, β3A interacts both with the ubiquitously expressed μ3A and with the neuronal-specific μ3B (Fig. 3 c), and β3B interacts with μ3A (Fig. 3 d). For reasons that are not clear, we were unable to detect any interaction between β3B and μ3B (unpublished data). However, the observations that β3A can interact with μ3B and that β3B can interact with μ3A suggest that, instead of having a neuronal-specific complex and a nonneuronal-specific complex, the subunits can “mix and match” if they are coexpressed in the same cell. The interactions involving the AP-3 subunits appear to be complex-specific, as we could detect no interactions between β3 and μ1 (Fig. 3 c), or between δ and σ1 (unpublished data). Other pairs of subunits were also tested, but the only interactions we were able to detect were those shown in Fig. 3.


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

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

Subunit interactions between AP-3 subunits detected using the yeast two-hybrid system. Yeast cells were transformed with cDNAs in either pGBT9 or pGAD424, as indicated, and interactions were assayed by growth in the absence of histidine. Panels a and b show that the δ subunit interacts with both σ3A and σ3B, panel c shows that the β3A subunit interacts with both μ3A and μ3B, and panel d shows that the β3B subunit interacts with μ3A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199225&req=5

fig3: Subunit interactions between AP-3 subunits detected using the yeast two-hybrid system. Yeast cells were transformed with cDNAs in either pGBT9 or pGAD424, as indicated, and interactions were assayed by growth in the absence of histidine. Panels a and b show that the δ subunit interacts with both σ3A and σ3B, panel c shows that the β3A subunit interacts with both μ3A and μ3B, and panel d shows that the β3B subunit interacts with μ3A.
Mentions: To investigate interactions between the AP-3 subunits further, and also to determine whether the A and B isoforms of the various subunits can associate with each other, we cloned the cDNAs into the vectors pGBT9 and pGAD424 for analysis using the yeast two-hybrid system. Fig. 3 , a and b, shows that δ interacts under these conditions with σ3A and σ3B, both of which are expressed ubiquitously. In addition, β3A interacts both with the ubiquitously expressed μ3A and with the neuronal-specific μ3B (Fig. 3 c), and β3B interacts with μ3A (Fig. 3 d). For reasons that are not clear, we were unable to detect any interaction between β3B and μ3B (unpublished data). However, the observations that β3A can interact with μ3B and that β3B can interact with μ3A suggest that, instead of having a neuronal-specific complex and a nonneuronal-specific complex, the subunits can “mix and match” if they are coexpressed in the same cell. The interactions involving the AP-3 subunits appear to be complex-specific, as we could detect no interactions between β3 and μ1 (Fig. 3 c), or between δ and σ1 (unpublished data). Other pairs of subunits were also tested, but the only interactions we were able to detect were those shown in Fig. 3.

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