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. 2012 Jun 8;287(24):20625-35.
doi: 10.1074/jbc.M112.341487. Epub 2012 Apr 12.

Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome

Qihong Zhang  1 , Dahai Yu, Seongjin Seo, Edwin M Stone, Val C Sheffield
Affiliations

Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome

Qihong Zhang et al. J Biol Chem. .

Abstract

The pleiotropic features of obesity, retinal degeneration, polydactyly, kidney abnormalities, cognitive impairment, hypertension, and diabetes found in Bardet-Biedl syndrome (BBS) make this disorder an important model disorder for identifying molecular mechanisms involved in common human diseases. To date, 16 BBS genes have been reported, seven of which (BBS1, 2, 4, 5, 7, 8, and 9) code for proteins that form a complex known as the BBSome. The function of the BBSome involves ciliary membrane biogenesis. Three additional BBS genes (BBS6, BBS10, and BBS12) have homology to type II chaperonins and interact with CCT/TRiC proteins and BBS7 to form a complex termed the BBS-chaperonin complex. This complex is required for BBSome assembly. Little is known about the process and the regulation of BBSome formation. We utilized point mutations and null alleles of BBS proteins to disrupt assembly of the BBSome leading to the accumulation of BBSome assembly intermediates. By characterizing BBSome assembly intermediates, we show that the BBS-chaperonin complex plays a role in BBS7 stability. BBS7 interacts with BBS2 and becomes part of a BBS7-BBS2-BBS9 assembly intermediate referred to as the BBSome core complex because it forms the core of the BBSome. BBS1, BBS5, BBS8, and finally BBS4 are added to the BBSome core to form the complete BBSome.

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Figures

FIGURE 1.
FIGURE 1.
BBS2, BBS7, and BBS9 form the BBSome core complex. A, FLAG-tagged BBS7 containing a point mutation (T211I) and WT BBS7 were transfected into 293T cells. Both WT and mutant BBS7 were pulled down by FLAG beads. The existence of other endogenous BBSome subunits is detected by Western blot. B, the absence of BBS2 affects the interaction of BBS7 with BBS1, whereas the loss of BBS1 does not affect the interaction of BBS7 with BBS2. C, FLAG-tagged BBS5, BBS8, BBS9, and BBIP10 were transfected into 293T cells. Tagged proteins were immunoprecipitated (IP) by FLAG beads, and the existence of other endogenous BBSome subunits was detected by Western blot. D, BBS9 RNAi was used to knock down endogenous BBS9 expression in a stable cell line expressing FLAG-BBS2. The BBSome was immunoprecipitated by FLAG beads, and the existence of endogenous BBSome subunits was detected by Western blot.
FIGURE 2.
FIGURE 2.
BBS1 is incorporated into the BBSome before BBS4. A, Western blots of protein lysates from WT and Bbs1M390R/M390R mice demonstrate that the absence of BBS1 does not affect other BBSome subunits protein levels, whereas BBS1 protein levels are greatly decreased in samples from Bbs1M390R/M390R mice as indicated by the arrow. This antibody also recognizes several additional bands that do not change in intensity when compared between WT and Bbs1M390R/M390R mice, indicating that these bands are nonspecific. B, sucrose gradient analysis of BBSome formation in Bbs1M390R testes demonstrates that most BBS4 is dissociated from the subcomplex consisting of BBS2, BBS5, BBS7, BBS8, and BBS9. C, immunoprecipitation (IP) using antibody against BBS2 from testes protein lysates shows that BBS4 is dissociated from the subcomplex in the absence of BBS1. These results are similar to those shown by sucrose gradient analysis. D, reciprocal immunoprecipitation from transfected 293T cells demonstrates that BBS4 dissociation from BBS1M390R is due to decreased interaction between BBS1M390R and BBS4 compared with WT BBS1. E, diagram showing the sequential order of BBS1 and BBS4 incorporation into the BBSome.
FIGURE 3.
FIGURE 3.
BBS4 is likely the last subunit to incorporate into the BBSome. A, sucrose gradient analysis of BBSome formation in Bbs4−/− testes lysates demonstrates that all other BBSome subunits can still form a complex in the absence of BBS4. B, immunoprecipitation using anti-BBS2 antibody from testes protein lysates indicates that BBS4 is the last subunit to incorporate into the BBSome. C, diagram showing the sequential order of BBS components incorporation into the BBSome.
FIGURE 4.
FIGURE 4.
BBS4-PCM1 interaction is required for the BBSome to localize to centrosome/pericentriolar satellite. A, BBS4 point mutations (G277E and R295P) disrupt the interaction between BBS4 and the C terminus of PCM1. GFP-tagged BBS4 (WT or mutants) and HA-tagged PCM1 C terminus were co-transfected into 293T cells, and co-immunoprecipitation (IP) was performed using antibody against HA and GFP. B, BBS4 point mutations (G277E and R295P) disrupt centrosome/pericentriolar satellite localization of BBS4. Shown is immunofluorescent staining of GFP-BBS4 (WT and mutants) in transfected RPE1 cells. γ-Tubulin (red) is used as a centrosomal marker. C, kidney primary cells were transfected with GFP-BBS4, and transfected cells were co-stained with γ-tubulin. The bar represents 10 μm.
FIGURE 5.
FIGURE 5.
BBS10 is required for the interaction of BBS6-BBS12-BBS7 with the CCT/TRiC proteins to form the BBS-chaperonin complex. A, BBS10 RNAi is used to knock down the expression of BBS10 in 293T cells expressing FLAG-BBS6, which was used to pull down the BBS-chaperonin complex. Antibodies against BBS7 and the CCT/TRiC proteins were used to detect the existence of these proteins in the complex. B, overexpression of BBS10 facilitates the association of BBS6 with the CCT/TRiC proteins. FLAG-BBS10 was co-transfected with empty vector or Myc-BBS6, and Myc antibody was used to pull down the BBS-chaperonin complex. Antibodies against BBS7, TCPα, and TCPβ (two subunits of the CCT/TRiC proteins) were used to detect these proteins in the complex. IP, immunoprecipitation.
FIGURE 6.
FIGURE 6.
The release of BBS7 from the BBS-chaperonin complex is coordinated with BBSome core complex formation. A, FLAG-BBS6 was overexpressed in 239T cells to trap BBSome intermediates. We pulled down BBS6 using FLAG beads and detected endogenous BBSome subunits by Western blot. FLAG-BBS5 was used as a positive control, and FLAG-GFP was used as a negative control. B, individual BBSome subunits were overexpressed in 293T cells and were used to determine specific associations with the CCT/TRiC proteins. Only BBS7 and BBS2 can associate with CCT/TRiC proteins. FLAG-VHL was used as a positive control. FLAG-GFP and FLAG-BBS3 were used as negative controls. IP, immunoprecipitation.
FIGURE 7.
FIGURE 7.
BBS2 is ubiquitinated and degraded by the proteasome. A, the stability of BBS2 is dependent on the BBSome core complex, as well as the BBS-chaperonin complex. Proteins from BBS knock-out mouse tissue (testis) or from RNAi-treated cell lysates were separated by SDS-PAGE. Anti-BBS2 antibody was used to detect BBS2 protein levels. B, BBS2 is sensitive to protease treatment. Primary cells from WT and Bbs6−/− kidney were infected with adenovirus expressing FLAG-BBS2. Equal amounts of total proteins were mixed with protease thermolysin at 4 °C for the indicated time. Compared with WT cells, BBS2 degrades faster in Bbs6−/− cells, suggesting that BBS2 is not stable in the absence of BBS6. C, 293T cells and FLAG-BBS2 stable-expressing cells were treated with the proteasome inhibitor MG132. BBS2 was immunoprecipitated (IP) by FLAG beads or anti-BBS2 antibody, and ubiquitinated BBS2 was detected by anti-ubiquitin antibody. D, HA-ubiquitin was transfected into 293T cells, and endogenous BBS2 was immunoprecipitated by anti-BBS2 antibody. Ubiquitinated BBS2 was detected by anti-HA antibody or anti-ubiquitin antibody. E, 293T cells were treated with 50 μg/ml of cycloheximide (Chx) with or without 20 μm proteasome inhibitor MG132 or MG115 for 8 h. Endogenous BBS2 in the cell lysates was detected by anti-BBS2 antibody.
FIGURE 8.
FIGURE 8.
Intrinsic protein-protein interactions and chaperonin mediated sequential assembly of the BBSome. Shown are diagrams of our current model for BBSome assembly. BBS7 is stabilized by the BBS-chaperonin complex (BBS6-BBS12-BBS7-CCT) and makes a transition to the BBSome core complex (BBS7-BBS2-BBS9). This transition requires BBS10. The intrinsic protein-protein interactions guide the incorporation of BBS1, BBS5, and BBS8. BBS4 requires BBS1 to incorporate into the complex and is the last subunit to be added to the complex.
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