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. 2017 Nov 15;6:e27434. doi: 10.7554/eLife.27434

Figure 1. Subcomplexes of the BBSome.

(A) Coomassie-stained SDS-PAGE of binary and multimeric BBSome complexes purified by Strep/Flag tandem affinity purification. The BBS proteins are indicated with color-coded marks. Variances in gel mobility of the individual components is caused by different affinity tags, as is most pronounced for BBS18. (B) Schematic subunit organization model that is consistent with expression and stability of subcomplexes (Components of the most stable core complex in color, and the two missing subunits in grey).

Figure 1.

Figure 1—figure supplement 1. Coomassie-stained SDS-PAGE on individual BBSome subunits.

Figure 1—figure supplement 1.

(A) Cell lysates of baculovirus-infected Hi5 insect cells expressing the BBS proteins 1–12 (three days post-infection). For BBS1, both the full length (FL) and a C-terminally truncated version (residues 1–430) were tested, and for BBS7 two different transcript variants (t1 and t2) are shown. (B) Purified BBS subunits after single-step Streptactin-affinity purification.
Figure 1—figure supplement 2. Normalized gel filtration profiles of different BBS complexes.

Figure 1—figure supplement 2.

The samples were purified by affinity chromatography and subsequent gel filtration, and 2.5–10 µg of the peak fraction was then analyzed on a Superdex 200 5/20 column. The complexes containing 4, 5 and 6 subunits are composed of BBS 4,8,9,18; BBS 1,5,8,9,18 and BBS 1,4,5,8,9,18, respectively. Their elution volumes were plotted against the theoretical molecular weight of the monomeric complex (black circles) and compared with standard proteins (open grey circles, and a grey line for the linear fit line). The elution volume of the C-terminal domain of BBS9 fits better to a dimeric species, which is shown as a red circle.
Figure 1—figure supplement 3. Purification of BBSome complexes.

Figure 1—figure supplement 3.

(A) Gel filtration profile of BBSome complexes with 6 and 8 subunits. As can be seen in coomassie-stained SDS-PAGE of the gel filtration (GF) fractions (B), the affinity-purified core BBSome complex (six subunits) elutes as a monomeric species, while BBS2 and BBS7 only partially incorporate in the full eight subunit complex that elutes as an oligomeric species, separated from the 6mer. (C) Calibration of the gel filtration profile of the core BBSome with a standard mixture of proteins with known molecular weight. (D): The apparent molecular weight of the core BBSome (large sphere), as determined by a comparison with standard proteins (small squares) is slightly larger than its theoretical molecular weight. (E) Coomassie-stained SDS-PAGE of samples from the triple-affinity purification of the core BBSome complex. After initial Streptactin-affinity (Strep) and Nickel affinity (Ni) purifications, Prescission Protease (PP) was added to cleave off the tag on BBS18. After incubation for 12 hr at 4°C, aggregates were removed by gel filtration, and the pooled monomeric complex (GF) was further purified in a final Flag affinity purification step (E: Elution, FT: Flowthru). The three tags used for purification are a cleavable dual-Strep tag on BBS18 (18 s), a His-tag on BBS4 (4 hr) and a Flag-tag on BBS8 (8 F).