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. 2002 Jan 21;156(2):327-36.
doi: 10.1083/jcb.200107140. Epub 2002 Jan 21.

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

Andrew A Peden  1 , Rachel E Rudge, Winnie W Y Lui, Margaret S Robinson
Affiliations

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

Andrew A Peden et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
Immunoprecipitates and Western blots of AP-3 subunits from mh and pe cells. Extracts of cultured mh cells (left), cultured pe cells (middle), and spleens from both pe and control mice (right) were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions, with the exception of the anti-μ3 immunoprecipitations, which were performed in the presence of SDS. The gels were then blotted onto nitrocellulose and probed with the indicated antibody. An antibody against the AP-1 γ subunit was included as a control. The mh cells can be seen to express trace amounts of β3 and μ3, which form a dimer, whereas the σ3 subunit appears to be monomeric. The δ and σ3 expressed in the pe cells also form a dimer, and in addition a small amount of complete heterotetramer is made. The various subcomplexes that form in the mh and pe cells are indicated diagrammatically.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 8.
Figure 8.
Functional rescue of pe cells by the various β3 constructs. (a) A mixture of cells transfected with empty vector and cells expressing β3A were incubated 6 h with anti–LAMP-1 directly conjugated to Alexa Fluor 488, then double labeled with anti-δ. β3A-expressing cells (arrow), which could be identified by the punctate, perinuclear distribution of δ, took up less anti–LAMP-1 than cells transfected with empty vector. (b) Anti–LAMP-1 uptake analyzed by flow cytometry. The control peak shows the fluorescence intensity of a β3A-expressing cell line incubated with a control antibody (anti-KLH) conjugated to Alexa Fluor 488. The empty-vector peak shows the level of fluorescence intensity when a nonexpressing pe cell line was incubated with the anti–LAMP-1 antibody, whereas the β3A peak shows the level of fluorescence intensity when the pe cell line expressing β3A was incubated with the anti–LAMP-1 antibody. Anti–LAMP-1 uptake in the β3A-expressing cell line was 27% that of the empty vector–transfected line. (c) Graph of the results obtained by flow cytometry. In each case three different cell lines, stably expressing each of the six constructs were analyzed, as well as three cell lines transfected with empty vector. In each experiment, 10,000 cells were counted and the experiments were repeated on different days and the results for each cell line were averaged. The values were obtained by dividing the geometric mean fluorescent intensity of cells incubated with the anti–LAMP-1 antibody by that of cells incubated with the control antibody. Error bars show the standard deviation from the mean. β3A, β3B, and β3A817AAA constructs all gave good rescue (P ≤ 0.0001); β3Aβ2 and β3AΔ807–831 both gave partial rescue (P = 0.0059 and P = 0.0015, respectively); and β3A807 stop gave no significant rescue (P = 0.2523). P values were obtained using a one-tailed unpaired t test. Bar, 20 μm.
Figure 5.
Figure 5.
(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.
Figure 6.
Figure 6.
Immunofluorescence of cell lines expressing each of the six constructs labeled with anti-δ. All of the cells show punctate labeling with some concentration in the perinuclear region, indicating that the AP-3 complexes assembled from all six constructs are recruited onto membranes. Bar, 20 μm.
Figure 7.
Figure 7.
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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