Fig 2: TSPAN5 interacts with AP4 and forms a complex with GluA2 and Stargazin.(A) GST-pulldown experiment on adult rat hippocampus and cortex lysates using empty GST or GST fused to TSPAN5 C-terminus (GST-Ct). Input: 2.5% of pulldown volume. Blots probed for AP4ε. Red Ponceau shows the GST-bound fragments. (B) Co-immunoprecipitation experiment on adult rat hippocampus and cortex lysates. Input: 2.5% of the immunoprecipitated volume. Immunoprecipitation: α-rabbit IgG, α-TSPAN5, α-AP4σ, or α-AP4ε. Blots probed for TSPAN5, AP4σ, and AP4ε. (C) GST-pulldown experiments on adult rat hippocampus and cortex lysates using empty-GST or GST fused to the C-terminus of TSPAN5 (GST-Ct). Input: 2.5% of pulldown volume. Blots probed for GluA2/3, GluA1, Stargazin, and NMDAR subunit GluN2A. n = 3 independent experiments. Unpaired Student T test. (D) Quantification of experiment in panel C: intensity of the pulldown band for GluA2/3 and GluA1 each normalised on their input (pulldown/input: GluA2, 1.09±0.07; GluA1, 0.63±0.09). (E) GST-pulldown experiments on adult rat hippocampus and cortex lysates using empty-GST or GST-fused to the C-terminal of Stargazin (GST-Ct Stargazin). Input: 2.5% of pulldown volume. Blots probed for GluA2/3, TSPAN5, and CD81.Values represent the mean ± SEM. *=p < 0.05, **=p < 0.01, ***=p < 0.001. Figure 2—source data 1.List of the proteins identified with the yeast two-hybrid screening performed with full-length or C-terminal tail of TSPAN5 and individual data values for the bar graphs in panel D. Figure 2—source data 2.Raw images and images with cropped areas highlighted of the blots in panels A, B, C, and E.

Fig 3: TSPAN5 regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) exocytosis through recycling endosomes by the formation of a tetrameric complex with AP4 and Stargazin.Working model of TSPAN5 function in mature neurons (left) and TSPAN5 silencing effects (right). TSPAN5 forms a complex with Stargazin and AMPARs in the endoplasmic reticulum or in endoplasmic reticulum-Golgi intermediate compartment (ERGIC) vesicles. The presence of TSPAN5 is necessary to direct the GluA2 and Stargazin complex to the plasma membrane. TSPAN5 silencing in neurons induces the redirection of GluA2-containing vesicles to lysosomal degradation.