FIGURE 1.

Isolation of Tepsin as an AP-4 interactor. A, schematic representation of adaptor protein (AP) complexes, indicating their subunits and structural domains. Two other complexes not represented here, the COPI-F subcomplex and part of the TSET complex, have a similar structure. Notice that some ear domains comprise two subdomains referred to as sandwich and platform, while others consist of only a sandwich (e.g. AP-1γ) (16, 17) or platform (e.g. AP-4 β4) (41) subdomain. In addition, GGA proteins have a sandwich domain similar to the AP-1 γ-ear domain (18, 19) and the yeast exomer Chs5 subunit has a FBE domain that is structurally homologous to the sandwich-platform tandem of the AP-2 α-ear domain (50). B, top, consensus secondary structure prediction of tepsin by a combination of the MLRC, DSC, and PHD methods at the NPS@ server. Blue represents α-helix, red β-sheet, and magenta random coil structure. B, bottom, domain organization of human tepsin. Domain names and amino acid numbers are indicated. C, filtered results of the affinity purification and mass spectrometric analysis for proteins that co-purify with the human AP-4 complex. D, yeast two-hybrid (Y2H) analysis of the interaction of tepsin or p53 (control) fused to the GAL4 DNA binding domain (BD) with different AP-4 β4 constructs or the SV40 large T antigen (T-Ag) (control) fused to the GAL4 transcriptional activation domain (AD). Colonies were grown in the presence of varying concentrations of 3-amino-1,2,4-triazole (AT) to test the specificity and strength of the interactions. Colony growth in medium without histidine (−His) is indicative of interactions. Growth in complete medium (+His) is used as a control. All images are from plates grown for 4 days. The larger diameter of the growth areas on plates with higher concentrations of AT (particularly at 1–5 mm AT) stems from the fact that the yeast inoculum (5 μl drop) spreads over a larger area in these plates.