Fig. 5.

TMIGD1 interacts with SYNJ2BP. a Schematic presentation of murine Synj2bp isoforms. The TMIGD1-interacting fragment isolated from the yeast-two hybrid library (indicated in red) encodes AA 1–105 of Synj2bp and encompasses the entire PDZ domain (AA 13–100). Abbreviations: TM, transmembrane, MT-IM, mitochondrial intermembrane (b) TMIGD1 interacts with SYNJ2BP through its PDZ domain-binding motif. Lysates of SYNJ2BP-transfected HEK293T cells were incubated with GST-fusion proteins containing the entire cytoplasmic domain of TMIGD1 (GST-TMIGD1) or the cytoplasmic domain lacking the PDZ domain-binding motif (GST-TMIGD1/Δ5) or with GST alone (GST-). Precipitates obtained with GST fusion proteins were immunoblotted with anti-SYNJ2BP antibodies (Sigma #HPA000866, top panel, 90% of precipitates) or with anti-GST antibodies (bottom panel, 10% of precipitates). SYNJ2BP precipitated with GST-TMIGD1 but not with GST-TMIGD1/Δ5. Abbreviations: IB, immunoblotting; Lys, lysate. c SYNJ2BP co-immunoprecipitates with TMIGD1. Immunoprecipitates obtained with anti-TMIGD1 polyclonal antibodies (Affi1611) from HEK293T cells transfected with empty vector (pFlag vector), Flag-TMIGD1 and/or Flag-SYNJ2BP as indicated were immunoblotted with anti-SYNJ2BP antibodies (Sigma #HPA000866, left panel, 90% of precipitates) or with anti-Flag antibodies (right panel, 10% of precipitates). Note that SYNJ2BP is present in TMIGD1 immunoprecipitates. Abbreviations: endog., endogenous; IB, immunoblotting; IP, immunoprecipitation; Lys, lysate. d SYNJ2BP localizes at mitochondria in sparsely seeded cells but to cell-cell contacts in confluent cells. HK-2 cells were either sparsely seeded or grown to confluency for 3d, 7d, or 12 d as indicated, then fixed and stained for SYNJ2BP and either Mitotracker or ZO-1 as indicated. Scale bars: 10 μm