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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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(a) Schematic diagrams of six β3-based constructs that were stably transfected into pe cells. (b) Extracts from cell lines expressing each of the six constructs, as well as a cell line stably transfected with empty vector, were immunoprecipitated with anti-δ, and blots were probed with antibodies against δ, the construct itself (in most cases anti-β3A, although the last two lanes were probed with antibodies against β3B and β2, respectively), μ3, and σ3. Only δ and σ3 can be detected in the immunoprecipitate from the cell line transfected with the empty vector, whereas all four subunits can be detected in the cell lines expressing the six constructs, indicating that complete heterotetramers have been formed.
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fig5: (a) Schematic diagrams of six β3-based constructs that were stably transfected into pe cells. (b) Extracts from cell lines expressing each of the six constructs, as well as a cell line stably transfected with empty vector, were immunoprecipitated with anti-δ, and blots were probed with antibodies against δ, the construct itself (in most cases anti-β3A, although the last two lanes were probed with antibodies against β3B and β2, respectively), μ3, and σ3. Only δ and σ3 can be detected in the immunoprecipitate from the cell line transfected with the empty vector, whereas all four subunits can be detected in the cell lines expressing the six constructs, indicating that complete heterotetramers have been formed.

Mentions: The availability of an assay for AP-3–mediated sorting means that we can now test whether not only wild-type subunits, but also subunits that have been altered in some way, are functional. Because some such subunits might be expected to give intermediate rescue, we developed a quantitative assay based on FACS analysis for use on stably transfected cells (see below). Unfortunately, we have so far been unable to obtain any mh cell lines that were homogeneously expressing δ; thus, we decided to concentrate on pe cells. In addition to cells expressing wild-type β3A, we have established pe cell lines expressing β3B, a β3A-β2 chimera containing the β3A NH2-terminal domain (amino acids 1–686) but the β2 hinge and ear domains (β3Aβ2); two β3A deletion mutants (β3AΔ807–831 and β3A807 stop); and a β3A point mutant (β3A817AAA). The β3AΔ807–831 mutant is missing a fragment of the distal part of the hinge, including the clathrin binding domain (amino acids 817–822), whereas the β3A807stop mutant is missing the distal part of the hinge, including the clathrin binding domain, and all of the ear. In the β3A817AAA mutant, the clathrin binding motif has been mutated from SLLDLD to AAADLD. This same mutation has been shown to abolish clathrin binding in vitro (Dell'Angelica et al., 1998; unpublished data). Schematic diagrams of the constructs are shown in Fig. 5 a.


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

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

(a) Schematic diagrams of six β3-based constructs that were stably transfected into pe cells. (b) Extracts from cell lines expressing each of the six constructs, as well as a cell line stably transfected with empty vector, were immunoprecipitated with anti-δ, and blots were probed with antibodies against δ, the construct itself (in most cases anti-β3A, although the last two lanes were probed with antibodies against β3B and β2, respectively), μ3, and σ3. Only δ and σ3 can be detected in the immunoprecipitate from the cell line transfected with the empty vector, whereas all four subunits can be detected in the cell lines expressing the six constructs, indicating that complete heterotetramers have been formed.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: (a) Schematic diagrams of six β3-based constructs that were stably transfected into pe cells. (b) Extracts from cell lines expressing each of the six constructs, as well as a cell line stably transfected with empty vector, were immunoprecipitated with anti-δ, and blots were probed with antibodies against δ, the construct itself (in most cases anti-β3A, although the last two lanes were probed with antibodies against β3B and β2, respectively), μ3, and σ3. Only δ and σ3 can be detected in the immunoprecipitate from the cell line transfected with the empty vector, whereas all four subunits can be detected in the cell lines expressing the six constructs, indicating that complete heterotetramers have been formed.
Mentions: The availability of an assay for AP-3–mediated sorting means that we can now test whether not only wild-type subunits, but also subunits that have been altered in some way, are functional. Because some such subunits might be expected to give intermediate rescue, we developed a quantitative assay based on FACS analysis for use on stably transfected cells (see below). Unfortunately, we have so far been unable to obtain any mh cell lines that were homogeneously expressing δ; thus, we decided to concentrate on pe cells. In addition to cells expressing wild-type β3A, we have established pe cell lines expressing β3B, a β3A-β2 chimera containing the β3A NH2-terminal domain (amino acids 1–686) but the β2 hinge and ear domains (β3Aβ2); two β3A deletion mutants (β3AΔ807–831 and β3A807 stop); and a β3A point mutant (β3A817AAA). The β3AΔ807–831 mutant is missing a fragment of the distal part of the hinge, including the clathrin binding domain (amino acids 817–822), whereas the β3A807stop mutant is missing the distal part of the hinge, including the clathrin binding domain, and all of the ear. In the β3A817AAA mutant, the clathrin binding motif has been mutated from SLLDLD to AAADLD. This same mutation has been shown to abolish clathrin binding in vitro (Dell'Angelica et al., 1998; unpublished data). Schematic diagrams of the constructs are shown in Fig. 5 a.

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