Identification of a sorting motif for Tspan3 to MHCII compartments in human B cells

Tetraspanins are four-transmembrane proteins that play fundamental roles in the immune system by enabling processes like migration, proliferation, signaling and protein trafficking. While the importance of cell surface tetraspanins has been established, the function of intracellular tetraspanins is less well understood. Here, we investigated the role of tetraspanin 3 (Tspan3) in lymphocytes. Tspan3 expression was low in T cells and high in B cells which increased during B cell activation. Tspan3 localized to late endosomal structures where it colocalized with major histocompatibility complex II (MHCII) in the MHCII compartment. There, inhibiting the formation of intraluminal vesicles (ILVs) showed that Tspan3 localization was not affected in contrast to its homologue CD63. Using a peptide-pulldown approach, we identified that Tspan3 interacts with AP2 via its C-terminus that harbors a YXXΦ-based sorting motif. Interestingly, mutating this motif did not impair Tspan3 localization. Instead, leucine 246 was required for its intracellular localization, identifying an unrecognized leucine-based sorting motif responsible for Tspan3 localization to MHCII compartment in B cells. Taken together, we report a new sorting motif for Tspan3 to MHCII compartments in human B cells.