(A–C) The affinity of the 4mer BBSome complex containing BBS4, 8, 9 and 18 (A) and of the core BBSome complex containing BBS1, 4, 5, 8, 9, 18 (B) to different lipids were probed in a protein-lipid overlay assay. For this, hydrophobic membranes with immobilized lipids as depicted in (C) (so-called ‘PIP-strips’, Echelon) were blocked with TBS-T + 3% fatty acid–free BSA and then incubated with 7.5 µg/ml complex for one hour at room temperature. After washing three times with TBS-T + 3% fatty acid–free BSA, immobilized complexes were detected by Western blot against the Flag-tag on BBS8. The PIP strip experiments indicate that BBSome subcomplexes interact specifically with PIPs even in the absence of both the Arl6-binding subunit BBS1 and the previously described PIP-binding subunit BBS5 (40). (D): Potential orientation of the BBSome core complex towards the membrane. The orientation of Arl6 towards the BBSome was deduced from the crystal structure of the β-propeller of BBS1/Arl6 (Mourão et al., 2014), which was overlaid with the BBS1 β-propeller from the BBSome core complex. In such an arrangement, a positively charged surface of the core complex would be oriented towards the membrane (E,G) and a negatively charged cleft in the vicinity of BBS1 is favorably positioned to accept BBSome-binding regions from cargo proteins like GPCRs (E–G), which were found to be mostly positively charged (Klink et al., 2017). A model how GPCRs might be recognized by the BBSome is depicted in (G).