Assembly and function of AP-3 complexes in cells expressing mutant subunits.
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.
Peden
Andrew A
AA
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.
Rudge
Rachel E
RE
Lui
Winnie W Y
WW
Robinson
Margaret S
MS
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D005434:Flow Cytometry; D051907:Lysosomal Membrane Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence; D034041:Monomeric Clathrin Assembly Proteins; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D033942:Adaptor Proteins, Vesicular Transport; D015703:Antigens, CD; D002352:Carrier Proteins; D002966:Clathrin; D051907:Lysosomal Membrane Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; C439023:clathrin assembly protein AP180; C018589:lysosomal proteins