Deletion of a YEDL sequence in SLC35D3 perturbs its targeting to platelet dense granule precursors

Solute carrier family 35 member D3 (SLC35D3) is an integral membrane protein required for platelet dense granule (DG) biogenesis. SLC35D3-null mice display defective DG formation and prolonged bleeding time, yet the underlying mechanism remains unclear. Proper targeting of SLC35D3 to DGs is crucial for their physiological function. Here, we identified the Y³¹¹EDL sequence located in the C-terminal tail of SLC35D3, as a key determinant for its sorting into DG precursors. Deletion of this motif resulted in the accumulation of SLC35D3 on the plasma membrane. Using immunoprecipitation and pulldown assays, we demonstrated that the Y³¹¹EDL deletion also impaired SLC35D3 binding to the adaptor protein complex-3 (AP-3), although substitution of Y³¹¹EDL with alanines did not disrupt sorting or AP-3 binding. Moreover, we found that the interaction between SLC35D3 and the biogenesis of lysosome-related organelles complex 1 (BLOC-1) was dependent on AP-3 for endolysosomal targeting. We also showed that AP-3 and BLOC-1 deficiencies led to the lysosomal degradation of SLC35D3. This likely explains the observed reduction in SLC35D3 levels in platelets from AP-3 or BLOC-1 deficient mice. Together, our findings suggest that the Y³¹¹EDL sequence is required for the proper intracellular sorting of SLC35D3 to platelet DG precursors, thereby ensuring their functions.