AP-3 is a heterotetrameric adaptor involved in the biogenesis of lysosome-related organelles. The function of AP-3 as an adaptor relies on its ability to bind to membranes in an Arf-dependent fashion and to recognize sorting signals in the cytosolic tails of the transmembrane cargo. Here, we report an interdomain interaction involving the ear domain of the δ subunit and the σ3 subunit of AP-3. This interaction interferes with the binding of AP-3 to Arf but not to dileucine-based sorting signals. As a consequence, the δ-ear inhibits the recruitment of AP-3 to membranes both in vitro and in vivo and impairs the sorting of lysosomal membrane proteins. These observations suggest a new regulatory mechanism for the recruitment of AP-3 to membranes involving δ-ear-σ3 interactions.
