A dileucine motif in TMEM163 is essential for its binding with both AP-3 and BLOC-1 complex

) in TMEM163 is essential for interactions with BLOC-1 and AP-3 but dispensable for binding with AP-1, AP-2 and BLOC-2. Mutation of this motif led to TMEM163 accumulation in the plasma membrane. Loss of either BLOC-1 or AP-3 enhanced TMEM163 bound to the other and exhibited differential abnormal endo-lysosomal localization, suggesting competitive binding during TMEM163 trafficking. Collectively, our findings established TMEM163 as a cargo protein sequentially sorted by AP-3 and BLOC-1 via a shared dileucine-based sorting signal, which is essential for its proper trafficking to platelet DGs, uncovering a unique mechanism in HPAC cargo sorting and LRO biogenesis.