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

Abstract

The mouse mutants mocha and pearl are deficient in the AP-3 δ and β3A 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 β3 and μ3 subunits coassemble into a heterodimer, whereas the σ3 subunit remains monomeric. In pearl cells, the δ and σ3 subunits coassemble into a heterodimer, whereas μ3 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 β3 and μ3 can interact with each other. Pearl cell lines were generated that express β3A, β3B, a β3Aβ2 chimera, two β3A deletion mutants, and a β3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only β3A, β3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The β3Aβ2 chimera and the β3A short deletion mutant gave partial functional rescue, whereas the β3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of β3 are important for function, but the clathrin binding site is not needed.