Limits...
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.

Show MeSH

Related in: MedlinePlus

Phenotype of rescued and nonrescued mh cells. mh cells were transiently transfected with wild-type δ, then incubated with a mixture of rat anti–LAMP-1(b) and fluorescent WGA (c) for 6 h. Triple labeling shows that the cell in the field that is expressing δ has taken up less anti–LAMP-1 than its neighbors, indicating that there is less misrouting of LAMP-1 to the plasma membrane in this cell. Uptake of WGA is similar in all of the cells. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199225&req=5

fig4: Phenotype of rescued and nonrescued mh cells. mh cells were transiently transfected with wild-type δ, then incubated with a mixture of rat anti–LAMP-1(b) and fluorescent WGA (c) for 6 h. Triple labeling shows that the cell in the field that is expressing δ has taken up less anti–LAMP-1 than its neighbors, indicating that there is less misrouting of LAMP-1 to the plasma membrane in this cell. Uptake of WGA is similar in all of the cells. Bar, 20 μm.

Mentions: Previous studies have shown that the steady-state distribution of lysosomal membrane proteins, such as LAMP-1, LIMP-2, and CD63, appears normal in AP-3–deficient cells, but that they follow different trafficking routes to the lysosome. Le Borgne et al. (1998) demonstrated that cells that had been depleted of μ3 by incubation with antisense oligonucleotides showed increased endocytosis of antibodies against LAMP-1 and LIMP-2 that had been added to the culture medium, indicating that these proteins had been rerouted to the cell surface. Dell'Angelica et al. (1999)(2000) reported similar findings on primary cultures of fibroblasts from β3A-deficient patients and from mh and pe mice. We have also found that antibodies against LAMP-1 are readily endocytosed by both the mh and the pe cell lines (see Figs. 4 and 8) . However, because all cell lines are different from each other, we needed to confirm that the antibody endocytosis was a direct result of the AP-3 deficiency. Thus, we transiently transfected the cells with a wild-type copy of the defective subunit. Cells transfected with both the δ and the β3A constructs could be identified with a δ-specific antibody: the nontransfected mh cells were unlabeled, whereas the nontransfected pe cells gave diffuse rather than punctate labeling with the anti-δ antibody (see Fig. 1). Fig. 4 shows transiently transfected mh cells labeled for δ (a), endocytosed anti-LAMP-1 (b), and endocytosed WGA (c). The cell expressing δ has taken up significantly less anti–LAMP-1 than its nontransfected neighbors, while all of the cells have taken up similar amounts of WGA, indicating that endocytosis in general is not impaired in the δ-expressing cell. Similar results were obtained with pe cells transfected with β3A (see Fig. 8 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)

Phenotype of rescued and nonrescued mh cells. mh cells were transiently transfected with wild-type δ, then incubated with a mixture of rat anti–LAMP-1(b) and fluorescent WGA (c) for 6 h. Triple labeling shows that the cell in the field that is expressing δ has taken up less anti–LAMP-1 than its neighbors, indicating that there is less misrouting of LAMP-1 to the plasma membrane in this cell. Uptake of WGA is similar in all of the cells. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Phenotype of rescued and nonrescued mh cells. mh cells were transiently transfected with wild-type δ, then incubated with a mixture of rat anti–LAMP-1(b) and fluorescent WGA (c) for 6 h. Triple labeling shows that the cell in the field that is expressing δ has taken up less anti–LAMP-1 than its neighbors, indicating that there is less misrouting of LAMP-1 to the plasma membrane in this cell. Uptake of WGA is similar in all of the cells. Bar, 20 μm.
Mentions: Previous studies have shown that the steady-state distribution of lysosomal membrane proteins, such as LAMP-1, LIMP-2, and CD63, appears normal in AP-3–deficient cells, but that they follow different trafficking routes to the lysosome. Le Borgne et al. (1998) demonstrated that cells that had been depleted of μ3 by incubation with antisense oligonucleotides showed increased endocytosis of antibodies against LAMP-1 and LIMP-2 that had been added to the culture medium, indicating that these proteins had been rerouted to the cell surface. Dell'Angelica et al. (1999)(2000) reported similar findings on primary cultures of fibroblasts from β3A-deficient patients and from mh and pe mice. We have also found that antibodies against LAMP-1 are readily endocytosed by both the mh and the pe cell lines (see Figs. 4 and 8) . However, because all cell lines are different from each other, we needed to confirm that the antibody endocytosis was a direct result of the AP-3 deficiency. Thus, we transiently transfected the cells with a wild-type copy of the defective subunit. Cells transfected with both the δ and the β3A constructs could be identified with a δ-specific antibody: the nontransfected mh cells were unlabeled, whereas the nontransfected pe cells gave diffuse rather than punctate labeling with the anti-δ antibody (see Fig. 1). Fig. 4 shows transiently transfected mh cells labeled for δ (a), endocytosed anti-LAMP-1 (b), and endocytosed WGA (c). The cell expressing δ has taken up significantly less anti–LAMP-1 than its nontransfected neighbors, while all of the cells have taken up similar amounts of WGA, indicating that endocytosis in general is not impaired in the δ-expressing cell. Similar results were obtained with pe cells transfected with β3A (see Fig. 8 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