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.

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

“…5), indicating that γ1 and γ2 are mostly expressed as part of an AP-1 complex. In fact, it has been previously demonstrated that in the absence of one of the subunits, the respective AP complex becomes unstable and disassembles, resulting in decreased levels of the other subunits Meyer et al, 2000;Motley et al, 2003;Peden et al, 2002). Moreover, as for γ1, interaction of Nef with γ2 absolutely requires co-expression of the σ1 subunit (Mattera et al, 2011;and our results, data not shown).…”

Two patients with Hermansky Pudlak syndrome type 2 and novel mutations in AP3B1

“…2,5 7,12-15 Previous studies have shown that absent β3A leads to instability and rapid degradation of the entire AP-3 complex. 2,16 The AP-3 mutations in this study also result in loss of the entire AP-3 complex, even when the truncated 80kDa form of β3A, encoding the N-terminal trunk and part of the hinge, is expressed.…”

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

“…They mediate the sorting of membrane proteins among numerous intracellular compartments (1)(2)(3)(4). Importantly, they aid in the vesiculation of donor membranes through direct association with membrane cargo and other vesicle-coat proteins (5).…”

“…Four different types of adaptor protein have been identified (AP-1-AP-4) (1, 2, 4). These adaptor proteins are heterotetrameric complexes composed of a large variable subunit (uniquely named for each AP complex), a second large subunit (␤ subunit), a medium-sized subunit (referred to as ), and a small subunit ( subunit) (2,5,6). Although a few APs sort the same or similar cargo, some of the specificity of different AP functions may arise from their selective recruitment to the correct membrane compartment (3,5).…”

“…In mocha mice, the complete loss of all forms of AP-3 leads to defects in neurological and hematopoietic cell function (14); these animals exhibit balance problems and deafness, and they are hyperactive and undergo seizures (14,15). Selective ablation of ubiquitous AP-3 observed in pearl mice results in animals with defects in hematopoietic cells but no neurological symptoms (2) [with the possible exception of night blindness (16)]. Animals lacking either neuronal AP-3 subunit, ␤3B (17) or 3B (5, 9), exhibit neurological deficits that are less severe than those in mocha animals.…”