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Supplemental Information

Intrinsic protein-protein interaction mediated and chaperonin assisted sequential assembly of a stable Bardet Biedl syndome protein complex, the BBSome *

Qihong Zhang1#, Dahai Yu1, 3#, Seongjing Seo1, Edwin M. Stone2, and Val C. Sheffield1

Supplementary Materials and Methods Antibodies and Reagents

Rabbit polyclonal antibodies against human BBS1, BBS2, BBS4, and BBS7 have been previously described. All antibodies were affinity purified and the specificities were verified in knockout mice or by RNAi in cell culture. Monoclonal anti-β-tubulin, polyclonal anti-β-actin, monoclonal anti-acetylated tubulin, monoclonal anti-γ-tubulin, rabbit anti-BBS8 and BBS9, thermolysin, and cycloheximide were purchased from Sigma (St. Louis, MO). Goat anti-BBS2 (C-16) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat anti-TCP1α and TCP1β were purchased from Stressgen Biotechnologies Inc (San Diego, California). Rabbit anti-ubiquitin antibody was from Abcam (Cambridge, MA). Smartpool RNAi against human BBS2, BBS10, BBS12, BBS9 were from Dharmacon (Lafayette, CO). MG132 and MG115 were purchased from Calbiochem (San Diego, CA). Cell culture, transfection, and Immunofluorescence microscopy

Skin fibroblast cells from a human patient with the common BBS10 mutation, c91fs95 and control skin fibroblast cells were purchased from Coriell Institute (Camden, New Jersey) and cultured in DMEM high glucose with 10% FBS. 293T cells were cultured in DMEM high glucose with 10% FBS, hTERT-RPE1 cells were cultured in DMEM/F-12 medium with 10% serum. All tansfections were performed using lipofectamine 2000 or RNAiMAX (Invitrogen, Carlsbad, CA). For RNAi transfection, a final concentration of 50-100nM RNAi was used. For immunofluorescence, cells were fixed in cold methanol for 5 min at -200C and were washed with 3 x PBS and blocked with blocking buffer (1% BSA in PBS). Primary antibodies were diluted in blocking buffer and incubated at room temperature for 1 hour. Cells were washed with 3 x PBS, blocked with blocking buffer, then incubated with Alexa 488 or Alexa 568 labeled secondary antibodies (Invitrogen, Carlsbad, CA). The slides were mounted with Vectashield mounting media containing DAPI (Vector Laboratories, Burlingame, CA). Immunoprecipitation

Differentially tagged human BBS genes were co-transfected into 293T cells. Forty-eight hours after transfection, the cells were lysed in lysis buffer (1 x PBS, 1% Triton X-100, and protease inhibitor, Roche, Indianapolis, IN) and spun at 20,000 x g for 15 min at 40C. The supernatants were cleared by incubation with protein G beads (Pierce, Rockford, IL). Cleared lysates were incubated with antibodies against corresponding tags for 2 hrs. Protein G beads were then added and incubated for another 2 hrs. The beads were washed four times with lysis buffer and the interactions were detected by Western blotting.

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Western blotting Wild type and mutant testes were disrupted by TISSUEMISER (Fisher Scientific, Pittsburgh, PA) in lysis buffer (1 x PBS, 1% Triton X-100 and protease inhibitor, Roche, Indianapolis, IN). The disrupted tissues were freeze-thawed three times. The lysates were then centrifuged at 20,000 x g for 15 min and the concentrations of the supernatants were measured using the Bio-Rad Dc protein assay (Hercules, CA). Proteins were separated by electrophoresis using 4-12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) followed by transfer to nitrocellulose membranes and were detected by SuperSignal Dura extended substrate (Pierce, Rockford, IL). Site-directed mutagenesis

Homozygote point mutations were generated by site-directed mutagenesis (Stratagene, La Jolla, CA) and verified by DNA sequencing. Supplementary Figure Legends Supplemental Figure S1. Summary of Homozygote point mutations identified in BBS patients, which were used in this study. Supplemental Figure S2. BBS7 interacts with BBS9 through BBS2. A. BBS7 strongly interacts with BBS2 and weakly with BBS1. Shown are co-IP assay of Flag-BBS7 with myc-tagged other BBSome subunits in 293T cells. B. BBS7 interacts with BBS9 through BBS2. Shown are co-IP assay of Flag-BBS7 with myc-BBS9 and HA-BBS2 in 293T cells. Supplemental Figure S3. Summary of the BBS4 binding domains for PCM1 and BBIP10. A. BBS4 truncation mutants were generated by PCR. GFP was added to the N-terminal of each truncated protein. These GFP-BBS4 truncation mutants were co-transfected with HA-tagged PCM1 C-terminal fragment or myc-BBIP10. Co-IP results are summarized here. B. BBIP10 interacts with PCM1 only in the presence of BBS4. Supplemental Figure S4. BBS7, BBS6, and BBS12 form a subcomplex. A. The interaction of BBS6 with BBS12 requires BBS7. Shown are the co-IP assays of Flag-BBS6 with myc-BBS12 in the presence of control siRNA, BBS7 siRNA, or BBS2 siRNA. B. The interaction of BBS7 with BBS12 requires BBS6. Shown are co-IP assay of Flag-BBS12 with myc-BBS7 in the presence of control siRNA or BBS6 siRNA. C. Overexpression of WT BBS6 but not BBS6 mutants increases the interaction between BBS7 and BBS12. Supplemental Figure S5. Loss of BBS10 does not affect primary ciliogenesis. Shown are immunofluorescent staining of skin fibroblast cells from a BBS10 patient with the common c91fs95 mutation using anti-acetylated tubulin as a ciliary marker (green) and BBS9 (red). Similar percentages of cells contain cilia in WT and mutant cells. However, the intensity of BBS9 staining is greatly decreased in the BBS10 mutant cells.

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Supplemental Figure S6. Loss of BBS10 affects BBSome subunits protein levels and disrupts BBSome formation. A. Western blots of total protein from WT and BBS10c91fs95 skin fibroblast cells were probed with antibodies against BBSome subunits. Equal amount of proteins were loaded as demonstrated by BBS3, IFT88, and tubulin signals. B. Sucrose gradient analysis shows that loss of BBS10 disrupts BBSome formation. BBSome subunits are concentrated in fractions 12 and 13 of the sucrose gradient. BBSome subunits are totally absent in BBS10c91fs95 skin fibroblast cells. To demonstrate that the same amounts of proteins are loaded to the sucrose gradient, a non-specific band from BBS2 antibody is shown. C. Loss of BBS10 does not affect the BBSome subunit mRNA levels as shown by semi-quantitative RT-PCR. Supplemental Figure S7. BBS7 protein stability requires BBS6, BBS10, BBS2, and BBS9. A. Western blots of protein samples from Bbs2, Bbs6, Bbs4, and Bbs1M390R/M39R mutant mice testes, BBS10 mutant cell lines, or from BBS9 RNAi knocked-down RPE1 cells demonstrate that BBS7 protein stability requires BBS6, BBS10, BBS2, and BBS9, but not BBS1 or BBS4. B. BBS2 protein is not stable in the absence of BBS7. We introduced BBS2 into Bbs7 null kidney cells by adenovirus expressing Flag-BBS2. The stability of BBS2 was examined by the protease thermolysin sensitivity assay. The half-life of BBS2 in WT cells is significantly longer than in Bbs7 null cells.

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G141R, A455TBBS9

T211I, H323RBBS7

G72S, T183ABBS5

G277E, R295PBBS4

V75G, L125RBBS2

M390RBBS1

Homozygous point

mutations

Gene

Figure S1

5

IP: myc IB: FlagB

BS

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BB

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Figure S2

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51-519

236-519

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1-439

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1-269

BBS4

BBIP10 PCM1

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R295P

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Figure S3

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Figure S4

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ac-tub BBS9

contro

lBBS10c91fs95

Figure S5

9

BBS8

BBS1

BBS5

BBS4

BBS9

BBS7

BBS2

from BBS2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 3 4 5 6 7 8 9 10 11 12 13 14 15 16

control BBS10c91fs95

BBS1

BBS4

BBS2

BBS7

tubulin

total

WT

BBS10c91fs95

BBS3

BBS9

IFT88

BBS8

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control BBS10c91fs95

C

Figure S6

10

BBS7

tubulin

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Bbs6-/-

Bbs2-/-

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BBS10c91fs95

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Bbs1M390R

WT

Bbs4-/-

BBS7

tubulin

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Figure S7