A structure- based mechanism for initiation of AP-3 coated vesicle formation

Adaptor protein complex- 3 (AP- 3) mediates cargo sorting from endosomes to lysosomes and lysosome- related organelles. Recently, it was shown that AP- 3 adopts a constitutively open conformation compared to the related AP- 1 and AP- 2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP- 3 is regulated is therefore an open question. To understand the mechanism of AP- 3 membrane recruitment and activation, we reconstituted human AP- 3 and determined multiple structures in the soluble and membrane- bound states using electron cryo- microscopy. Similar to yeast AP- 3, human AP- 3 is in a constitutively open conformation. To reconstitute AP- 3 activation by adenosine di- phosphate (ADP)- ribosylation factor 1 (Arf1), a small guanosine tri- phosphate (GTP)ase, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures showing the stepwise conformational changes required for formation of AP- 3 coated vesicles. First, membrane recruitment is driven by one of two predicted Arf1 binding sites, which flexibly tethers AP- 3 to the membrane. Second, cargo binding causes AP- 3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP- 3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher- order coat structures. Additionally, we provide evidence for two amphipathic helices in AP- 3, suggesting that AP- 3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP- 3- mediated vesicle coat assembly.