BBSome is not formed in Bbs6 null mouse tissues. (A) Wild-type (WT) and Bbs6 null (6KO) mouse testis extracts were fractionated by size exclusion chromatography. Elution fractions were subjected to SDS/PAGE and immunoblotting. Membranes were probed with antibodies against Bbs1, Bbs2, Bbs4, Bbs7, and Bbs8. (B) Extracts from wild-type and Bbs6 null eye were analyzed as in A. Open arrowhead indicates Bbs1; closed arrowhead indicates a cross-reacting protein. It should be noted that Bbs1 blot from Bbs6 null eye was exposed longer than that of wild-type.

Requirement of CCT activity for BBSome assembly, (A) CCT chaperonins are required for BBSome assembly. HA-BBS9 was cotransfected with siRNAs against CCT1, CCT2, CCT3, or control in HEK293T cells, and BBS9-associated BBSome subunits were probed by immunoprecipitation followed by immunoblotting. (B) Chaperonin-like BBS proteins are required for the interaction between BBS7 and CCT chaperonins. Expression of BBS6, BBS10, and BBS12 was blocked by RNAi in HEK293T cells, and endogenous BBS7 was immunoprecipitated in control, BBS6-, BBS10-, and BBS12-depleted cells. Association of CCT chaperonins with BBS7 was measured by immunoblotting. (C) MO-mediated knockdown of CCT chaperonins results in Kupffer’s vesicle (KV) defects in zebrafish. Representative images of zebrafish embryos and KVs are shown at Left. (Top) Lateral view of a 10-somite stage embryo with the location of the KV circled, dorsal views; (Middle and Bottom) of a normal KV (arrow) and a reduced KV (arrowhead) at the 10-somite stage. Embryos were injected with 15 ng of indicated MOs, except for cct1, for which a 5-ng dose was used because of severe developmental defects and lethality. The percentage of embryos with reduced or absent KVs are represented for each MO treatment. Sample size and P values are in Table S2. **P < 0.01 determined by Fisher’s exact test compared with control MO-injected group. (D) Retrograde melanosome transport is delayed in cct2 morphants and genetic mutants. Melanosome transport is assessed by monitoring melanosome trafficking in the head region of zebrafish (Top). Below are representative images of the boxed area with dispersed melanosomes of dark-adapted zebrafish before epinephrine treatment (Middle) and contracted melanosomes after epinephrine induced transport (Bottom). Rate of melanosome transport to the endpoint (perinuclear distribution) is noted in seconds on y-axis. Data are represented as mean ± SEM. **P < 0.01, determined by one-way ANOVA and Tukey test. (Scale bar, 100 μm.)

Identification of the BBS/CCT complex. (A) Interactions between BBS6, BBS10, and BBS12. Myc- or FLAG-tagged BBS6, BBS10, and BBS12 expression constructs were transfected into HEK293T cells as indicated and lysates were subjected to co-IP assay. (Middle and Bottom) Expression of each component in the lysate. (Top) Amounts of FLAG-tagged proteins coimmunoprecipitated by anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting. Open arrowhead indicates IgG heavy chain. (B) BBS6, BBS10, and BBS12 associate in vivo. HEK293T cells were transfected with 1 μg of FLAG-BBS12 in one 10-cm dish and immunoprecipitated with anti-FLAG antibody. Co-IP of endogenous BBS6 and BBS10 was probed by Western blotting using antibodies against BBS6 and BBS10. β-Actin was used for normalization of the input. (C) Purification of BBS6 and BBS12 containing protein complexes. Proteins from Myc-BBS6– and FLAG-BBS12–expressing cells (BBS6+12) were purified by sequential affinity purification and analyzed by SDS/PAGE and silver staining. Parental cells were used as a control. Size markers (M) are shown in the left and IgG heavy chain (H) and light chain (L) are marked. (D) Copurification of BBS10 and BBS2 as minor interacting proteins of the BBS/CCT complex. Proteins isolated by sequential purification were resolved in SDS/PAGE and immunoblotted for BBS10 and BBS2. (E) Chaperonin-like BBS proteins form a multisubunit complex with CCT chaperonins. Proteins partially purified by anti-FLAG affinity gel were fractionated by Superose-6 size exclusion chromatography. Elution volumes and approximate molecular weights of fractions where BBS proteins were found were denoted at Top. Void volume was ∼7.6 mL. Fractions were immunoblotted for Myc (BBS6), FLAG (BBS12), BBS10, BBS7, CCT1, CCT2, and CCT3.

Chaperonin-like BBS proteins are required for BBSome assembly. (A) BBSome assembly in BBS6-, BBS10-, and BBS12-depleted cells. HA-BBS9 was transfected into control, BBS6-, BBS10-, and BBS12-depleted HEK293T cells, and BBSome assembly was assessed by measuring BBSome subunits associated with BBS9. (B) BBS2 deletion mutant constructs used to map BBS6- and BBS7-interacting domains. Open diamonds represent FG-GAP motifs (FG); black box indicates the coiled-coil (CC) domain; and gray box indicates the C-terminal α-helix–rich domain. Numbers represent amino acid residues. (C) Coiled-coil domain in BBS2 interacts with BBS6. HA-tagged BBS2 deletion mutants were cotransfected with FLAG-BBS6 into HEK293T cells, and lysates were immunoprecipitated with anti-HA antibodies. (D) C-terminal domain in BBS2 interacts with BBS7, but BBS6-interacting domain is required for efficient binding with BBS7. GFP-BBS7 was cotransfected with BBS2 deletion mutants.

Many disease-causing missense mutations found in BBS6, BBS10, and BBS12 disrupt interactions among these proteins. (A) Interactions of BBS6 missense mutants with BBS12. HEK293T cells were transfected with Myc-BBS12 together with HA-BBS6 variants and lysates were subject to coimmunoprecipitation (IP) and immunoblotting (IB). Numbers at bottom represent ratio of coprecipitated proteins compared with wild-type protein after normalization with input. (B) Interactions of BBS10 missense mutants with BBS12. (C) Interactions of BBS12 missense mutants with BBS6. (D) Model for BBS/CCT complex function. BBS/CCT complex initially binds to BBS7 and potentially to BBS2, and mediates their association with other BBSome subunits (BBS1,4,5,8,9) to assemble BBSome. When chaperonin-like BBS genes are inactivated, at least two BBSome subunits (BBS2 and BBS7) are degraded, and the remaining BBSome subunits exist in monomeric form or aggregates with unidentified proteins.