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

Defects in Hermansky-Pudlak syndrome protein associated complexes impair the biogenesis of lysosome-related organelles (e.g. platelet dense granule). Transmembrane protein 163 (TMEM163), a zinc transporter, is drastically reduced in platelets of AP-3-, BLOC-1-, and BLOC-2-deficient Hermansky-Pudlak syndrome mice and patients, but the mechanistic basis is unclear. We here found that TMEM163 was reduced and degraded primarily via the proteasome in a variety of deficient MEG-01 cells of endosomal trafficking complexes (AP-1, AP-2, AP-3, BLOC-1, BLOC-2). A conserved N-terminal acidic dileucine motif (LEDRGL69L70) 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 dense granules, uncovering a unique mechanism in Hermansky-Pudlak syndrome protein associated complex cargo sorting and lysosome-related organelle biogenesis.