Novel interaction partners of Bardet-Biedl syndrome proteins

Novel Interaction Partners of Bardet-BiedlSyndrome Proteins

F. Oeffner,* C. Moch, A. Neundorf, J. Hofmann, M. Koch, and K. H. Grzeschik

Center of Human Genetics, University of Marburg, 35037 Marburg, Germany

Bardet-Biedl syndrome (BBS) is a rare, developmental disorder characterized bysix major symptoms: rod-cone dystrophy, obesity, polydactyly, renal abnormal-ities, learning difficulties, and hypogonadism. Secondary features include cardiacand hepatic anomalies, metabolic disturbancies, and hearing loss. BBS is geneti-cally heterogeneous with 12 disease genes (BBS1–BBS12) described thus far. Cur-rent data suggest a functional disturbance in ciliary function and intraflagellartransport being associated with the phenotype. However, the precise functions ofthe BBS proteins have yet to be elucidated. This study focuses on the detection ofprotein factors interacting with BBS proteins. Applying yeast two-hybrid (Y2H)technology we found a series of novel, functionally potentially plausible bindingpartners of BBS1, BBS2, BBS4, and BBS7. Protein interactions were supportedby coimmunoprecipitation analyses (ALDOB, EPAS1) and substantiated bycolocalization studies at the subcellular level (ALDOB, EXOC7, FLOT1, KRT18,PAX2). Our work provides new insights into the understanding of BBS interac-tions and thus their biological function. Cell Motil. Cytoskeleton 65: 143–155,2008. ' 2007 Wiley-Liss, Inc.

Key words: Bardet-Biedl syndrome; protein interactions; yeast two-hybrid screening; centrosome; cilia

INTRODUCTION

The rare genetic disorder Bardet-Biedl syndrome(BBS) is a striking example of locus heterogeneity withat least 12 genes mapping to different chromosomal loci(BBS1–BBS12) associated with the disease. Thoughhaving only limited structural and functional similarities,mutant BBS proteins give rise to the same indistinguish-able phenotype [Green et al., 1989; Beales et al., 1999;Katsanis et al., 2001; Katsanis, 2004]. The cardinalsymptoms are retinal dystrophy, postaxial polydactyly,truncal obesity, renal dysplasia, learning difficulties, andhypogonadism with infertility in males. Additionally,BBS patients suffer from minor features at varying fre-quencies (e.g. anosmia, developmental delay, ataxia,hearing loss, diabetes mellitus, cardiovascular anoma-lies, liver disease).

The first three BBS genes identified (BBS6, BBS2,BBS4) share no structural similarities and were detectedby positional cloning [Katsanis et al., 2000; Slavotineket al., 2000; Mykytyn et al., 2001; Nishimura et al.,2001]. BBS6/MKKS is a group II chaperon-like protein,having evolved recently from a subunit of the cytosolic

chaperonin CCT [Kim et al., 2004]. BBS4 shows strong-est homology to O-linked N-acetylglucosamin transfer-ase (OGT) of several species. Some of the other BBSgenes have been identified via bioinformatic compari-sons of protein sequences and computational compara-tive genomics techniques: BBS1 has been detected bythe finding of limited sequence homology to BBS2[Mykytyn et al., 2002]. The cloning of BBS7 and BBS8

*Correspondence to: Frank Oeffner, Center of Human Genetics,

University of Marburg, Bahnhofstrasse 7a, 35037 Marburg, Germany.

E-mail: [email protected]

This article contains supplementary material available via the Internet

at http://www.interscience.wiley.com/jpages/0886-1544/suppmat.

Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract

grant number: OE 262/1-1.

Received 14 November 2006; Accepted 8 October 2007

Published online 13 November 2007 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cm.20250

' 2007 Wiley-Liss, Inc.

Cell Motility and the Cytoskeleton 65: 143–155 (2008)

was supported by database searches for sequences dis-playing partial homology to BBS2 and BBS4, respec-tively [Ansley et al., 2003; Badano et al., 2003]. BBS5,BBS3/ARL-6, and BBS9 were identified applying com-parative genomic analyses [Chiang et al., 2004; Fanet al., 2004; Li et al., 2004; Nishimura et al., 2005]. Thepositional cloning of BBS10 revealed a major BBS locus,mutated as frequently as BBS1 [Stoetzel et al., 2006]. Itcodes for a vertebrate-specific bona fide chaperonethat—contrary to BBS6/MKKS—has conserved its ATPbinding site. The last two BBS genes cloned till now—BBS11, encoding the E3 ubiquitin ligase TRIM32[Chiang et al., 2006] and BBS12, a further chaperonin-coding gene [Stoetzel et al., 2007]—have been identifiedby the means of high-density SNP genotyping and homo-zygosity mapping.

Among the 12 BBS proteins BBS1, 2, and 7 exhibitpartial structural similarity with each other, sharing apredicted b-propeller domain, while BBS6, 10, and 12are probably chaperonins. The first clues regarding ashared function for the heterogenous group of proteinswere revealed by the cloning of BBS8, which is expressedspecifically in cliliated structures, such as the connectingcilium of the retina or ciliated neurons in C. elegans. Onthe subcellular level it localizes to centrosomes and basalbodies and binds to PCM1, a protein presumably involvedin ciliogenesis [Ansley et al., 2003].

Since then, a functional overlap of BBS proteinsand ciliary/flagellar and centrosomal activities has beenwell established by studies undertaken in model organ-isms [Blacque et al., 2004; Mykytyn and Sheffield, 2004;Beales, 2005; Ross et al., 2005]. Several BBS homo-logues localize to basal bodies in C. elegans [Ansleyet al., 2003; Blacque et al., 2004; Fan et al., 2004; Liet al., 2004], where mutants of bbs7 and bbs8 show dis-turbancies of the intraflagellar transport (IFT) [Ansleyet al., 2003; Blacque et al., 2004]. According to Ou et al.the C. elegans IFT machinery likely has a modular archi-tecture with the BBS proteins stabilizing the motor-IFTparticle assembly [Ou et al., 2007].

Defects in the generation of flagella have beenshown in knock out mouse models of BBS2, BBS4, andBBS6, and also by RNAi silencing of BBS5 in Chlamy-domonas [Li et al., 2004]. Furthermore, gene knock outof Bbs2, Bbs4, and Bbs6 in mice seems to precludemaintenance of the photoreceptor and causes mislocationof rhodopsin [Mykytyn et al., 2004; Nishimura et al.,2004; Fath et al., 2005]. BBS4 is a pericentriolar proteinof centrosomes and also of basal bodies of primary cilia,where it binds to components of the dynein transport ma-chinery like PCM1 and the dynactin-dynein microtu-bule-based molecular motor [Kim et al., 2004]. Thus,BBS seems to entail the dysfunction of the primary cil-ium. BBS proteins are assumed to assist in the mainte-

nance of cilia and/or the survival of some ciliated cells.Furthermore, there is growing evidence that BBS pro-teins play a general role in intracellular trafficking byassisting microtubule-related (retrograde) transport,which has been recently demonstrated in zebrafishknockdown models of bbs2, bbs4, bbs5, bbs6, bbs7, andbbs8 [Yen et al., 2006].

However, important questions regarding the pre-cise function and interactions of BBS proteins stillremain elusive. How do BBS proteins participate inmicrotubule-based intracellular transport processes?Which binding partners do individual BBS proteinsinteract with—and in this context—do they share cargolike BBS4 and BBS8, that interact with the same peri-centriolar protein PCM1? Do they interact to generate acommon multiprotein complex or do they influence/affect the same pathway sequentially?

To identify novel interaction partners of selectedBBS proteins (BBS1, BBS2, BBS4, BBS7) we adoptedthe yeast two-hybrid method. Binding partners of BBS1and BBS4, revealed by screening a human fetal kidneycDNA library, were tested for interaction with BBS2 andBBS7 (short isoform), as well. This approach enabled usto confirm the interactions of BBS4 with PCM1 andDCTN1 as described previously [Kim et al., 2004], andwe were able to identify additional promising bindingpartners (e.g. ALDOB, EPAS1, FHOD1, FLOT1, PAX2).Selected interactions—particularly between ALDOB andEPAS1 and BBS proteins analyzed in this study—wereindependently confirmed by coimmunoprecipitation. Asfor ALDOB, EXOC7, FLOT1, KRT18, and PAX2 wesuccessfully demonstrated subcellular colocalization withBBS proteins.

MATERIALS AND METHODS

Plasmids and Vector Construction

Plasmid Vectors. Vectors pGBKT7, pGADT7,pGBKT7-Lam, pGBKT7-p53, pGADT7-T, pEGFP-C1,and pDsRed-Monomer-C1 were acquired from BD Bio-sciences (Heidelberg, Germany). Plasmid pGBKT7 con-tains the DNA binding domain (BD) of GAL4 fusedwith a c-myc epitope, a kanamycin resistance (KAN),and the TRP1 gene, whereas pGADT7 encodes the acti-vation domain (AD) of GAL4 combined with a hemag-glutinin (HA) epitope, an ampicillin resistance (AMP),and the LEU2 gene. The T7 promotor allows the synthe-sis of RNA by using T7 RNA polymerase and afterwardsthe in vitro-translation of cloned genes and gene frag-ments, respectively.

pEGFP-C1 encodes a red-shifted variant of wild-type green fluorescent protein (GFP), while pDsRed-Monomer-C1 codes for a monomeric mutant derived fromthe tetrameric Discosoma sp. red fluorescent protein.

144 Oeffner et al.

Recombinant Constructs. Vectors pGBKT7-BBS1, pGBKT7-BBS2, pGBKT7-BBS4, and pGBKT7-BBS7 were synthesized by fusing the coding sequenceof BBS1, BBS2 (short isoform), BBS4, and BBS7 withthe GAL4 binding domain of pGBKT7. Coding sequen-ces were either generated by reversely transcribinghuman fetal kidney RNA (BD Biosciences) (BBS1,BBS4) or by amplifying ORF clones purchased at theHuman Genome Resource Center (Berlin, Germany)(BBS2, BBS7). Applied primer sets are shown in Supple-mentary Table I online. Following standard PCR purifi-cation (QIAGEN: Hilden, Germany) DNA fragmentswere digested with suitable restriction endoucleases(BBS1/BBS2/BBS4: EcoR I, BamH I; BBS7: Nco I,BamHI) provided by Fermentas (Sankt Leon-Rot, Ger-many) and inserted into the multiple cloning site (MCS)of pGBKT7 in frame with the encoded c-myc/GAL4binding domain. Correct insertion of coding sequencesand absence of mutations was verified by sequencing theinserts on an automated DNA sequencer model 310(Applied Biosystems: Weiterstadt, Germany). TheMATCHMAKER 50DNA-BD Vector Insert ScreeningAmplimer (50-TCA TCG GAA GAG AGT AG-30) andthe 30DNA-BD Sequencing Primer (50-TAA GAG TCACTT TAA AAT TTG TAT-30) were provided by BDBiosciences and employed as sequencing primers.

Analogically, the coding sequences of the afore-mentioned BBS proteins and full-length coding sequen-ces of putative binding partners (ALDOB, EXOC7,FLOT1, KRT18, PAX2) were inserted into pEGFP-C1and pDsRed-Monomer-C1 to carry out cotransfection andimmunofluorescence studies. Applied primers are givenin Supplementary Tables II and III online, respectively.

Immunoblot Analysis

To verify the correct expression of the hybrid mol-ecules, protein extracts were isolated from yeast cells(strains: AH109, Y187) according to the urea/SDSmethod [Printen and Sprague, 1994] as described in theYeast Protocols Handbook (PT3024-1: BD Biosciences).Subsequently, yeast proteins resuspended in samplebuffer (12.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% so-dium dodecyl sulfate, 0.25% bromophenol blue, 5% b-mercaptoethanol) were boiled for 5 min and immediatelysubjected to discontinuous sodium dodecyl sulfate-9%polyacrylamide gel electrophoresis (SDS PAGE) andsemidry blotting using polyconal rabbit anti c-myc im-munoglobulin (BD Biosciences) and a peroxidase conju-gated anti rabbit secondary antibody (Sigma-Aldrich:Taufkirchen, Germany). Specific interactions betweenantigens and antibodies were visualized by the enhancedchemiluminescence system (ECL) (Amersham Bioscien-ces: Freiburg, Germany).

Yeast Two-Hybrid Analysis

We exploited the BD Matchmaker Two-HybridSystem 3 (BD Biosciences), since it provides four dis-tinct reporters (ADE2, HIS3, MEL1, and lacZ), therebyallowing for high stringency tests. To carry out two-hybrid screening in the budding yeast Saccharomycescerevisiae, plasmids pGBKT7-BBS1 and pGBKT7-BBS4, containing BBS1 and BBS4 fused to the DNAbinding domain of GAL4, were used as bait to screena human fetal kidney cDNA library (BD Bioscience)representing fusion proteins of the activation domainof GAL4. The library had been pretransformed by themanufacturer into yeast strain Y187 (MATa) and con-tained 3.5 3 106 independent clones. The screeningprocedure (handling of yeast cultures, plate growthassays, quantitative/qualitative b-galactosidase assays,and 3-AT competition experiments) to identify BBS1-and BBS4-interacting proteins was carried out inaccordance to the Matchmaker GAL4 Two-HybridSystem 3 & Libraries User Manual (PT3247-1: BDBiosciences).

Briefly, growth tests were usually performed underhigh stringency conditions in the absence of adenine andhistidine. Only interactions verified through these quad-ruple drop out (QDO) growth tests plus the filter lacZassay were regarded as true positives. Prey plasmidswere then isolated and sequenced as described above todetermine the identity of the putative BBS1 or BBS4binding partner using the MATCHMAKER 50 AD LD-Insert Screening Amplimer (50-CTA TTC GAT GATGAA GAT ACC CCA CCA AAC CC-30) and the 30 ADLD-Insert-Screening Amplimer (50-AAG TGA ACTTGC GGG GTT TTT CAG TAT CTA CG-30) assequencing primers. Sequences generated on the 310DNA sequencer were then searched against public data-bases using the BLAST program at the NCBI [Altschulet al., 1990]. Yeast colonies might also bear more thanone prey plasmid. To exclude the possibility of sequenc-ing the noninteracting prey construct, positive cloneswere individually cotransformed with the correspondingbait vectors (pGBKT7-BBS1/-BBS4) into yeast strainAH109 and then resequenced. A subsequent cotransfor-mation with pGBKT7-p53 served as falsification of falsepositives. Clones being inclined to interact with p53were excluded from further analyses.

A 30-AT inhibition assay was performed to quan-tify protein–protein interactions. 3-AT (3-amino-1,2,4-triazole) is a competitive inhibitor of the yeast HIS3 pro-tein (His3p). Merely clones showing strong interactionsbetween bait and prey will grow, whereas relativelyweak interactions are suppressed. Diploid cells weregrown for 1 week on QDO plates containing varying 3-AT concentrations from 2.5 to 30 mM.

Interacting Partners of BBS Proteins 145

Selected BBS1- and BBS4-interacting partners(encoded by the cDNA library plasmid pACT2) weretested in direct interaction assays using pGBKT7-BBS2and -BBS7 as bait. Interactions were verified applyingplate growth assays and qualitative/quantitative lacZassays in accordance to the manufacturer’s instructions(BD Biosciences: User manual PT3247-1).

In vitro Translation andCoimmunoprecipitation Assays

Due to the T7 promotor sequence, pGBKT7- andpGADT7-constructs can be applied directly in radioac-tive and nonradioctive in-vitro translation assays usingthe TNT

TM T7 coupled rabbit reticulocyte kit (Promega:Mannheim, Germany). pACT2 prey sequences first hadto be PCR amplified with a special primer set: pACT2-CoIP for (50-AAA ATT GTA ATA CGA CTC ACTATA GGG CGA GCC GCC ACC ATG TAC CCA TACGAT GTT CCA GAT TAC GCT-30) and pACT2-CoIP rev(50-ACT TGC GGG GTT TTT CAG TAT CTA CGAT-30). The T7 promotor in the forward primer is under-lined, whereas the characters in italics show the HA tagcoding region corresponding to nucleotides 5042–5068of pACT2. The translation start codon ATG is shown inbold characters. The reverse primer corresponds to nu-cleotides 4949–4976 of pACT2. Efficiency of in vitro-translation was determined by performing luciferase con-trol experiments in accordance with the manufacturer’sinstruction guide (Promega: TB126).

Radioactive coimmunoprecipitation experimentswere performed following the manufacturer’s instruc-tions (BD Biosciences: User manual PT3323-1) with theexception of a reduced number of washing steps (twosteps using wash buffer 1). Briefly, translated bait pro-teins (pGBKT7 constructs) were precipitated in the pres-ence of protein A beads, that recognized the FC part ofanti c-myc immunoglobulin bound by the c-myc tag. Inthe case of specific protein–protein-interactions the preyproteins are coimmunoprecipitated. Precipitated proteinswere resuspended in sample buffer containing 5% b-mercaptoethanol, boiled for 5 min and directly subjectedto 9% SDS-PAGE. After fixation (20% methanol, 10%acetic acid) for 40 min and treatment with amplify fluo-rographic reagent gels were vacuum-dried for 45 min.X-ray films were incubated at 2708C overnight anddeveloped the next morning.

Cell Lines and DNATransfection

HeLa (a human tumor cell line) cells were culturedin Dulbecco’s modified Eagle’s medium (DMEM) sup-plemented with 10% fetal calf serum plus penicillin (100U/ml) and streptomycin (100 lg/ml) and incubated at378C in 5% CO2. Plasmids pDsRed-BBS1/-2/-4/-7 and

pEGFP-ALDOB/-EXOC7/-FLOT1/-PAX2 were intro-duced using the nonliposomal EffecteneTM lipid trans-fection system (QIAGEN, Hilden, Germany).

Indirect Immunoflourescence Staining

Transfected cells were permeabilized and fixed byincubating in methanol acetone 1:1 for 1 min. Afterrehydration with multiple rinses in PBS, the cells weretreated with PBS containing 10% bovine serum albuminto block unspecific binding sites. Double immunofluores-cence staining of cells was carried out using a polyclonalrabbit anti g-tubulin antibody (T5192: Sigma-Aldrich)diluted 1:1000 in PBS containing 1.5% bovine serum al-bumin and 0.5% Tween 20. Washing steps were per-formed in PBS/0.5% Tween 20. An alexa fluor 594 goatanti rabbit immunoglobulin (Molecular probes) served assecondary antibody. Immunostained cells were againwashed thoroughly with PBS/0.5% Tween 20 andmounted in ProLong Antifade reagent containing DAPI(QIAGEN: Hilden, Germany). Photographs were takenusing a Zeiss Axioplan microscope (Zeiss: Oberkochen,Germany) equipped with a Leica DFC 300 FX camera(Leica Microsystems: Cambridge, England). Imageswere acquired and processed with the Leica imaging sys-tem 50 (IM50) software (Leica Microsystems).

RESULTS

Proteins From Human Fetal Kidney Cells InteractWith BBS1 and/or BBS4

To identify cellular factors that interact withselected BBS proteins (BBS1, BBS2, BBS4, BBS7 weapplied a stepwise approach: First, yeast two-hybridscreening was performed with BBS1 and BBS4 fused tothe C terminus of the DNA binding domain of GAL4(BD) as bait. A pretransformed cDNA library represent-ing fusion proteins of the transcription activation domainof GAL4 (AD) and cDNAs derived from human fetalkidney cells was screened. In preceding control experi-ments we proved that (a) the DNA-BD fusion constructswere not toxic for yeast cells, (b) did not activate re-porter genes autonomously, and (c) displayed the correctprotein expression pattern. BD-BBS1 showed theexpected size of 93 kDa (BD/c-myc: 22 kDa, BBS1: 71kDa), whereas the BD-BBS4 fusion protein ran at �100kDa (BBS4: 58 kDa), displaying additional minor frag-ments of 75, 52, and 35 kDa (data not shown).

After yeast mating diploid clones were grownunder high stringency conditions in the absence of tryp-tophane, leucine, adenine, and histidine resulting in 41(BBS1 screen) and 120 colonies (BBS4 screen), respec-tively. In parallel we calculated the mating efficienciesof both analyses to ensure that the number of clones

146 Oeffner et al.

screened was significantly higher than the number of in-dependent clones represented by the cDNA library (Sup-plementary Table IV online).

To isolate the pACT/prey plasmids diploid cellswere incubated in SD/-Leu selective medium thus expel-ling the bait constructs. Prey plasmids were isolatedfrom these colonies, transformed into E. coli and individ-ually cotransformed with the bait vectors pGBKT7-BBS1 and -BBS4 into yeast strain AH109. To verifypositive clones we afterwards performed highly stringentgrowth tests in QDO selective media and a qualitativefilter lacZ test thereby confirming all 41 clones of theBBS1 screen and 115 of the BBS4 analysis (data notshown). Prey plasmids were then cotransformed withpGBKT7-p53 as bait to allow for falsification of falsepositive clones. Having carried out QDO growth testsand filter lacZ tests we could finally confirm 39 (BBS1screen) and 102 (BBS4 screen) positive clones poten-tially interacting with BBS1 and BBS4 (data not shown).

To identify the prey proteins we performedsequencing analysis on the plasmid DNA purified fromE. coli. Sequences were compared with the GenBankdatabase of the NCBI via the basic local alignmentsearch tool (BLAST). Identified proteins were then di-vided in three groups (Table I): (a) Proteins being

observed repeatedly in the screening analyses (BBS1screen: ALDOB 7/39, TM4SF2 4/39, HSC20 andEEF1A1 3/39, and KRT18 2/39; BBS4 screen: ALDOB28/102, EIF3 6/102, PCM1 and FLOT1 3/102, EPAS1 2/102), (b) Proteins detected once in the screen seemingplausible binding partners due to the expression patternand putative biological function (BBS1 screen: DJ1;BBS4 screen: ACY, BHMT, CALCA, FHOD1, PAX2),and (c) Proteins detected once in the screen appearing asplausible binding partners due to the subcellular localiza-tion (BBS1 screen: EXOC7; BBS4 screen: ACTB,DCTN1).

In addition to growth tests and qualitative lacZanalyses all clones identified in the BBS1 screen weresubjected to a 3-AT competition assay. Cell growth wasassessed after 1 week incubation on 3-AT QDO plates.Most of the clones ceased growth at 2.5 mM. Two BBS1interactors (ALDOB, TM4SF2 still grew at 2.5 mM 3-AT, and one interacting partner (HSC20) survived at30 mM 3-AT.

Selected Proteins Interacting With BBS1 and BBS4Interact With BBS2 and BBS7, as well

Subsequently, we addressed the question whetherthese clones will interact also with BBS2 and BBS7.

TABLE I. Plausible Binding Partners of Selected BBS Proteins

Prey

pACT2- OMIM

Bait

pGBKT7-BBS- ORF

Expression/functional relevance in following tissues (organelles)

Kidney

Testis/

fertility Eye/retina Limbs CNS

Cilia/IFT,

vesicular

transport

Cyto-

skeleton

ACTBa 102630 4 0 45% X

ACYb 609924 1 2 4 7 30% X

ALDOBc 229600 1 2 4 7 49–66% X X

BHMTb 602888 1 2 4 0 80% X

CALCAb 114130 1 0 4 0 100% X

DCTN1a 601143 1 0 4 0 64–71% X

DJ1b 602533 1 0 0 0 40% X X

EEF1a1c 130590 1 2 4 0 65%

eIF3c 602039 1 0 4 0 55%

EPAS1c 603349 1 2 4 0 35% X X

EXOC7a 608163 1 2 4 0 50% X

FHOD1b 606881 1 2 4 7 60% X X X X

FLOTc 606998 1 2 4 7 70% X

HSC20c 608142 1 2 4 0 100%

KRT18c 148070 1 2 4 7 100% X

PAX2b 167409 1 2 4 7 60% X X

PCM1c 600299 4 0 55% X

TM4SF2c 300096 1 0 0 0 100% X

Clones identified by screening the pretransformed human fetal kidney cDNA libray with BBS1 and BBS4 as bait were classified into three

groups as follows:aClones detected repeatedly.bPlausible interactions due to expression and biological function.cPlausible interactions due to subcellular localization (e.g. cytoskelettal components, factors involved in vesicular transport). Interactions are

listed in the bait column given that QDO growth tests and filter lacZ assays had been positive. Fat formatted numbers indicate the analysis

(BBS1 or BBS4 screen) by which the prey protein was initially revealed. The extension of coding sequences found in both screens (from 30 ter-minus) is shown in column ORF.


Therefore, the coding sequences of BBS2 and BBS7 werecloned into pGBKT7, followed by sequencing analysisand the same control experiments as described above.Likewise BD-BBS4, they displayed degraded proteinfragments in addition to the correctly sized bands of 103kDa (BBS2: 81 kDa 1 22 kDa BD/c-myc) and 102 kDa(BBS7: 80 kDa 1 22 kDa BD/c-myc) (data not shown).Clones transformed into yeast strain Y187 served as preyfor pGBKT7-BBS2/-BBS4, and -BBS7 (interacting part-ners from the BBS1 screen) and for BBS1, BBS2, andBBS7 (binding partners from the BBS4 screen), respec-tively. After yeast mating, highly stringent QDO incuba-tion and filter lacZ analysis we observed interactionsamong 15 prey clones and at least two different BBSproteins (Table I). Six proteins were found to interactwith each of the BBS protein analyzed (ACY, ALDOB,FHOD1, FLOT1, KRT18, PAX2), five bound to BBS1/2/4 (BHMT, EEF1a1, EPAS1, EXO70, HSC20), threeprey clones represented binding partners of BBS1 andBBS4 (CALCA, DCTN1, eIF3), and four clonesappeared to interact with only one BBS protein (ACTB:BBS4, DJI: BBS1, PCM1: BBS4, TM4SF2: BBS1).Regarding PCM1 this was a reconfirmation of findingsalready described by Kim et al. [2004]. We found nosupport for interactions with one of the other BBS pro-teins in this study, whereas DCTN1 presumably alsointeracts with BBS1.

Since the pretransformed human kidney cDNAlibrary represents mainly protein fragments instead offull length constructs (Table I, column ORF), the entirecoding sequences of selected interactors (ALDOB,EEF1a1, PCM1) were cloned into pGADT7. Aftercotransformation with pGBKT7-BBS1/-BBS2/-BBS4/-BBS7 in yeast strain AH109, cells were subjected toQDO growth tests, filter lacZ analyses, and 3-AT compe-tition experiments. According to these experimentsALDOB again bound to all BBS proteins analyzed inthis study, however only weakly with BBS2 and BBS7(data not shown). Full length EEF1a1 and PCM1 pro-teins interacted with BBS1, BBS2, and BBS4. Theresults of experiments performed on full length clonesare summarized in Table II.

Interactions of ALDOB, EEF1A1, and EPAS1With BBS Proteins are Confirmed viaIn vitro Coimmunoprecipitation

Selected interactions detected through yeast-twohybrid methods were analyzed independently by in vitrocoimmunoprecipitation. The T7 promotor lying down-stream to the DNA-BD was used to transcribe and trans-late the c-myc-tagged BBS proteins (BBS1, BBS2,BBS4, BBS7). First, the correct in vitro translation of thefour BBS proteins analyzed in this work was verified(Fig. 1).

Radioactive in vitro translation of pGBKT7-p53and pGADT7-T followed by coimmunoprecipitationusing anti-c-myc-antibodies and protein A beads (BDBiosciences) served as positive control for the biochemi-cal verification of selected BBS protein-prey interactions(Fig. 2). Accordingly, the interaction between ALDOB(C-terminal moiety consisting of 164 amino acidresidues) and BBS1, BBS2, BBS4, and BBS7 has been

TABLE II. Quantification of Putative Interactions Between BBS1, BBS2, BBS4 and BBS7 and Selected Full Length Prey Proteins

QDO growth test Filter lacZ test 3-AT competition assay

BBS1 BBS2 BBS4 BBS7 BBS1 BBS2 BBS4 BBS7 BBS1 BBS2 BBS4 BBS7

ALDOB X X X X X X X X 5.0 0.2 10.0 1.0

EEF1a1 X X X X X X X 2.5 1.0 10.0

PCM1 X X X X X X 10.0 1.0 15.0

The positive outcome of either growth tests or filter lacZ assays is indicated with an ‘‘X’’. The 3-AT competition column displays the highest

molar concentrations of inhibitor with observable cell growth.

Fig. 1. In vitro translation and immunoprecipitation of BBS proteins.

The bait vectors encode the BBS proteins in fusion with the c-myc-

epitopes. The T7 promotor enables direct in vitro transcription fol-

lowed by radioactive translation. [35S]-tagged translations products

are then precipitated via c-myc antibodies and protein A beads,

respectively. Lane 1: c-myc-BBS1 (73 kDa), lane 2: c-myc-BBS2

(84 kDa), lane 3: c-myc-BBS4 (61 kDa), lane 4: c-myc-BBS7 (83 kDa).

148 Oeffner et al.

confirmed. Likewise, an EEF1a1 fragment (204/462 resi-dues) was coprecipitated with BBS1, whereas EPAS1(294/870 amino acids) was successfully precipitatedwith BBS4. These interactions, however, are relativelyweak, since the EPAS1 prey fragment was coelutedmerely with BBS4 as bait. No interactions were de-tectable in the case of EEF1a1/BBS7, HSC20/BBS1/2/4/7, DCTN1/BBS4, PCM1/BBS4, Thy1/BBS7, andTM4SF2BBS1/2/7.

Interactions of ALDOB, EXOC7, FLOT1, KRT18,and PAX2 With BBS Proteins are Supported byColocalization Studies

HeLa cells were transiently transfected with full-length ALDOB-, EXOC7-, FLOT1-, KRT18-, and PAX2-pEGFP constructs, while, g-tubulin—an establishedcentrosomal marker—was visualized using a polyclonalantibody. ALDOB and EXOC7 expression did not overlapwith g-tubulin but was detected in close vicinity to theMTOC, while FLOT1, KRT18, and PAX2 localize, at leastpartially, with g-tubulin within centrosomes (Fig. 3).

Additionally, HeLa cells were cotransfected withBBS1, BBS2, BBS4, and BBS7 inserted into pDsRedand ALDOB-/EXOC7-/FLOT1-/KRT18-/PAX2-pEGFP.Colocalization of BBS proteins and their interactionspartners was detectable at punctuate subcellular struc-tures, suggesting partially overlapping protein expressionat the microtubule-organizing center (Fig. 4).

DISCUSSION

Applying yeast two-hybrid technology, we havedetected several plausible binding partners of four Bar-det-Biedl syndrome proteins (BBS1, BBS2, BBS4,BBS7). These interactors include enzymes such as aldol-ase B (ALDOB), transcription factors like EPAS1 orPAX2, and proteins constituting components of the cyto-skeleton or participating in vesicular transport (KRT18).In addition to qualitative and quantitative (3-AT compe-tition assay) yeast two-hybrid experiments, the specificinteractions between selected binding partners and BBSproteins were supported by co-immunoprecipitation(ALDOB, EPAS1) and substantiated by colocalizationstudies performed with full length constructs (ALDOB,EXOC7, FLOT1, KRT18, PAX2).

The identification of BBS8 [Ansley et al., 2003]indicated anomalies in the assembly of basal bodiesand cilia as the central pathophysiological mecha-nism—a hypothesis which has also been confirmed forthe other BBS proteins [Ross et al., 2005]. Neverthe-less, the precise function of individual BBS proteinshas yet not been determined. Identifying interactors ofcertain BBS proteins might give a clue to additionalcellular activities not discovered so far and mayunravel subtle genotype-phenotype correlations. Fur-thermore, interactors of known BBS proteins may benovel BBS proteins—recent studies suggest the exis-tence of at least 17 BBS genes [Nishimura et al.,

Fig. 2. Interactions between al-

dolase B and selected BBS pro-

teins are confirmed by coimmuno-

precipitation. (A) Precipitation of

SV40 T antigen via anti-c-myc-

antibodies and A beads is accom-

plished in the presence of p53 and

serves as positive control. Lane 1:

HA-T, lane 2: c-myc-p53, lane 3:

HA-T 1 c-myc-p53. (B–D) Coim-

munoprecipitation of BBS proteins

and selected binding partners (B:

EEF1A1, C: EPAS1, D: ALDOB)

via anti-c-myc and protein A

beads. Lane 1: c-myc-BBS2, lane

2: C-myc-BBS protein 1 HA-

interacting partner, lane 3: HA-

interacting partner. The bait vec-

tors contained the complete coding

sequences of BBS1, BBS2, BBS4,

and BBS7, whereas the prey con-

structs encoded protein fragments.


2005; Laurier et al., 2006], and to improve diagnosticsand genetic counseling in BBS, it is necessary to iden-tify the remaining BBS genes.

To detect both relatively weak and transient pro-tein–protein interactions, we exploited a yeast-two-hybrid system that provided four distinct reporter genes,

Fig. 3. Association of full-length ALDOB, EXOC7, FLOT1, KRT18,

and PAX2 with centriolar satellites and the microtubule-organizing

center (MTOC). HeLa cells were transfected with pEGFP-ALDOB/-

EXOC7, -FLOT1, /-KRT18, /-PAX2 while the centrosome was stained

with an anti g-tubulin immunoglobulin. Nuclei were revealed by DAPI

staining. ALDOB and EXOC7 associate peripherally with the MTOC,

whereas FLOT1, KRT18, and PAX2 colocalize with g-tubulin in cen-

trosomes. Cells were imaged at 3100 magnification using oil immer-

sion on a Zeiss Axioplan microscope with a Leica DFF digital camera

using Leica imaging system 50 (IM50) software (Leica Microsystems).

150 Oeffner et al.

thereby also allowing for high stringency screening. Inour first set of experiments BBS1 and BBS4 were usedto screen a human fetal kidney cDNA library. BBS1 waschosen as bait because this locus contributes more than20% to BBS in Europeans [Katsanis, 2004] indicating arelative high medical relevance comparable to that ofBBS10 recently described by Stoetzel et al. [2006].

When commencing our analyses, BBS4 was supposed tobe mutated in almost 20% of affected individuals [Bru-ford et al., 1997], and it was among the first BBS genesto be cloned. However, other works clearly showed thatit is only a minor contributor to BBS with roughly 2% ofaffected persons displaying chromosomal inheritanceacross the BBS4 region [Katsanis, 2004]. Both of the

Fig. 4. Colocalization of full-length

ALDOB, EXOC7, FLOT1, KRT18, and

PAX2 with selected BBS proteins at subcel-

lular structures. Coimmunofluorescence stai-

ning in HeLa cells using full-length BBS1/

2/4/7 pDs-red constructs (red) and egfp

tagged ALDOB/EXOC7/FLOT1/KRT18/

PAX2 (green) for transient transfection.

Nuclei were counterstained with DAPI. The

cells were imaged as described for Fig. 3.


genes show strongest expression in the kidney [Mykytynet al., 2001, 2002]. Before screening the cDNA library,we demonstrated that BD-BBS1/-BBS4 hybrids were nottoxic for yeast cells and did not activate the ADE andHIS reporters autonomously. Protein expression wascontrolled with BD-BBS1 showing the expected size of93 kDa. However, the BD-BBS4 fusion protein ran at�100 kDa (expected: 58 kDa), also displaying severalminor gel fragments. The higher size of the major 100kDa fragment might be due to posttranslational modifi-cation in yeast, whereas minor fragments representcleavage products due to proteolytic activities. Since theamount of the higher molecular weight fragment sur-passed the degradation products, the BD-BBS4 chimerawas also used in screening analysis.

In parallel to the screening experiments leading to41 (BBS1 screen) and 120 (BBS4 screen) primary colo-nies, we calculated the corresponding number of clonesscreened. In both screens this experimental parameterexceeded the amount of 3.5 3 106 independent clones inthe cDNA library. Thus, the genetic information repre-sented by this library was screened repeatedly, therebyensuring that clones being expressed less frequentlywere not skipped.

Among the clones verified as true positivesALDOB was found with striking frequency—seventimes in the BBS1 screen and 28 times in the BBS4screen. Other proteins detected less frequently wereEEF1a1, HSC20, KRT18, TM4SF2 (BBS1 screen) andE1F3, EPAS1, FLOT, PCM1 (BBS4 screen). Clonesonly identified once might also be of biological signifi-cance due to their inferred function or subcellular local-ization, respectively. Plausibility and biological relevanceof these interactions will be discussed in detail below.

By carrying out yeast two-hybrid interaction assayswe also investigated whether those prey proteins inter-acting with BBS1 and BBS4 would bind to BBS2 andBBS7, as well. Among the BBS genes cloned so far, wedecided to take BBS2 as bait, because mutations in thisgene contribute to nearly 10% of BBS cases [Katsanis,2004]. BBS7 was chosen, because it cooperates withBBS1, BBS2, BBS4, and BBS6 in the so-called ‘‘trial-lelic inheritance’’ [Beales et al., 2003] suggestinggenetic interactions among these proteins in close sub-cellular vicinity. Among the BBS proteins analyzed,BBS7 showed up with a striking low frequency as bind-ing partner of different prey clones (6/19). Contrary toBBS7, the other BBS proteins were found with signifi-cant higher frequencies (BBS1: 17/19, BBS2: 12/19,BBS4: 17/19) indicating partially different binding part-ners of BBS7 as compared to those of BBS1, BBS2, andBBS4.

Among the prey proteins analyzed, ALDOB is oneof the most promising candidates. The finding that aldol-

ase B interacts with selected BBS proteins (BBS1/2/4/7)has been demonstrated for the first time by two inde-pendent methods: a yeast two-hybrid approach and coim-munoprecipitation analysis. It is well known that piecesof proteins might interact more unspecifically than fulllength clones. The prey proteins identified in the primaryBBS1 and BBS4 screens were represented by cDNAsfrom 0.4 (ATP1a1) up to 3 kB (EXOC7). To corroboratethose findings unraveled by the initial screening experi-ments, the whole coding sequence of ALDOB was like-wise subjected to yeast two-hybrid interaction assays. Inprincipal, interactions between ALDOB fragments (180/364 amino acid residues) and BBS1/2/4/7 were con-firmed using the full length protein as prey. Usually, invitro methods are employed to confirm true interactionssuggested by two-hybrid results. To circumvent the in-terference from coeluted antibody fragments (25 and50 kDa bands in reducing SDS-PAGE gels) possiblyobscuring the results, we performed a radioactive coim-munoprecipitation with selected prey proteins, such asALDOB. Subsequently, the interaction between anALDOB fragment (representing the C-terminal moietyof 180 residues) with BBS1/2/4/7 was successfully dem-onstrated.

In colocalization assays ALDOB expression didnot overlap with g-tubulin, but was found adjacent to thecentrosome, possibly associated in part to the centriolarsatellites (Fig. 3). This observation suggests a specificmechanism excluding ALDOB from the centrioles. Aftercotransfection of HeLa cells, ALDOB and BBS proteins(BBS1, BBS2, BBS4, BBS7) colocalized at punctuatesubcellular structures, presumably representing themicrotubule-organizing center (Fig. 4), thereby support-ing the experimental outcome of our Y2H analysis.

Recent studies support the biological relevance ofour findings by implicating aldolase in vesicular andmolecular transport [Buscaglia et al., 2006] activities inwhich also BBS proteins presumably participate [Blacqueand Leroux, 2006; Yen et al., 2006]. Thus our work is inline with studies of other groups that suggest numerousunrelated binding partners of aldolase, thereby attributingnovel ‘‘moonlighting’’ activities in cellular physiology inaddition to its well-known role in carbohydrate meta-bolism [Weed et al., 2000; Copley, 2003]. Furthermore,aldolase has not been mentioned as a typical false positivein the publicly available databases.

EXOC7 (exocyst complex component) plays a rolein protein transport and exocytosis, regulating togetherwith Sec6 and Sec8 the compartmentalization of Glut4-containing vesicles in adipocytes [Inoue et al., 2006] andcontrolling insertion of AMPA-type glutamate receptorsat the postsynaptic membrane in the CNS [Gerges et al.,2006]. EXOC7 was visualized both at the trans-Golginetwork and centrosomes suggesting a functional impli-

152 Oeffner et al.

cation in vesicular trafficking [Xu et al., 2005]. Theexocyst complex has also been found to modulatemicrotubule dynamics underlying exocytosis [Wanget al., 2004]. Similar to ALDOB, EXOC7 was detectedin close vicinity to, but not directly attached with, thecentrosomal centrioles (Fig. 3). Likewise, cotransfectionexperiments revealed overlapping expression patternsof BBS proteins analyzed in this study and EXOC7at punctuate subcellular sites—putative centrosomes(Fig. 4).

KRT18, belonging to the intermediate filaments, isa cytoskeletal component shown to interact with BBS1/2/4/7. As for the likely association of BBS proteins inmicrotubular dependent vesicular transport it appears asa plausible candidate genes. However, one must keep inmind, that keratin-related proteins also show up often asfalse positives in yeast two-hybrid experiments (e.g.www.fccc.edu/research/labs/golemis/main-false.html).For that reason the same cotransfection assays werecarried out as described for ALDOB and EXOC7 to cor-roborate our hypothesis of KRT18 as a BBS bindingcomponent of the cytoskeleton. As it is shown in Figs. 3and 4, colocalization of egfp tagged KRT18 with g-tubu-lin and BBS1/2/4/7 was demonstrable at centrosomes ofHeLa cells.

A further putative binding partner associated withvesicular trafficking is FLOT1, a member of the flotillinsthat constitute parts of an clathrin-independent endocy-totic pathway [Frick et al., 2007]. Colocalization assaysrevealed the same overlapping expression with g-tubulinand BBS proteins at centrosomes as shown for KRT18(Figs. 3 and 4).

Endothelial PAS domain protein 1 (EPAS1) is atranscription factor belonging to the PAS (Per-ARNT-Sim) family. Knock-out mice without EPAS1 activityshowed retinal degeneration and azoospermia [Scorte-gagna et al., 2003]—traits overlapping with BBS symp-toms. EPAS1 was found twice in the BBS4 screen andlater on found to interact with BBS1 and BBS2, as well.These interactions, however, were relatively weak, sincean EPAS1 prey fragment (294/870 amino acid residues)was coeluted merely with BBS4 as bait.

PAX2 is a further transcription factor presumablyinteracting with all BBS proteins analyzed in this work.It is expressed during the development of the eye, thekidney, the ear, and the CNS [Sanyanusin et al., 1995].PAX2 deficient individuals suffer from compromised re-nal function and progressive visual impairment—clinicalsymptoms that remind us of BBS. Alike KRT18, PAX2expression overlapped with g-tubulin and BBS1/2/4/7 incentrosomes. The disturbance of direct interactionsbetween BBS proteins and this morphogenetic factorcould account for at least some of the renal tissue abnor-malities in BBS patients.

The EEF1a1/BBS1 interaction shown by yeast-twohybrid analysis and coimmunoprecipitation might be oflimited biological relevance. EEF1a1 (eukaryotic trans-lation elongation factor 1 alpha 1) belongs to a group ofproteins that have been already described as possiblefalse positives in yeast two-hybrid experiments. TheEEF1a1 fragment (204/462 amino acids) was merelycoprecipitated with BBS1, whereas the full-length pro-tein did not visibly coelute with neither BBS protein.This observation interestingly also applied to PCM1,which could not be precipitated together with BBS4 in avisible way—neither as C-terminal protein fragment noras full-length clone. These findings are explainable byseveral reasons: Certain proteins or fragments may notbe able to gain their active confirmation in the lysate mixused in this study. To visualize precipitated binding part-ners, radioactive [S35]-Met was introduced, and in somecases the coding sequence might not have containedenough Met residues. Some prey proteins (e.g. PCM1)overlap in size with their binding partners (BBS4),whose strong signals thus have obscured the preysignals.

The PCM1 protein is a centrosomal factor alreadyidentified as specific and plausible binding partner ofBBS4 and BBS8 [Ansley et al., 2003; Kim et al., 2004].Contrary to our primary screenings and interactionsassays, full length PCM1 seemed to interact with BBS1and BBS2 in addition to BBS4—however, yeast cellsbearing pGBKT7-BBS4 and pGADT7-PCM1 could sur-vive even at 15.0 mM 3-AT, indicating a relative stronginteraction compared to that with BBS1/2 (0.2 mM). Dif-ferences in the interaction pattern between protein frag-ments and their full-length counterparts are likely due tothe underpresentation of 50-interaction domains in thecDNA library. Thus, interactions between PCM1 andBBS1/2 are likely to be mediated through a differentbinding domain in the 50-moiety. On the other hand, wecould not reproduce the positive findings of the coimmu-noprecipitation experiment reported by Kim et al.[2004]; this is likely attributable to different experimen-tal procedures and/or technical complications during theprecipitation analysis. In this context we could also showan interaction between DCTN1—an established bindingpartner of dynein [Vaughan and Vallee, 1995], microtu-bules [Waterman-Storer et al., 1995], and also BBS4[Kim et al., 2004]—and BBS1. Both proteins PCM1 andDCTN1 have been ascribed vital roles in centrosomesand IFT [Dammermann and Merdes, 2002]; thus they areplausible binding partners of BBS proteins.

Fusion homology 2 domain containing protein 1(FHOD1) is a further prey protein interacting with allfour BBS proteins. It is structurally related to the forminsbeing implicated in limb and kidney development [Masset al., 1990]. Additionally, FHOD1 is associated with


cellular processes such as cytokinesis, cell polarity gen-eration and organelle-trafficking. As an heterologousmolecular motor it orchestrates actin filaments andmicrotubules [Gasteier et al., 2005; Bugyi et al., 2006;Schonichen et al., 2006].

HSC20 is a mitochondrial J-type cochaperone.Since diploid yeast cells containing pGBKT7-BBS1 andpGADT7-HSC20 still grew at 30.0 mM 3-AT, there isprobably a strong specific interaction with BBS1. How-ever, the biological relevance as far as BBS is concernedremains questionable, because as a heat-shock protein itbelongs to a group of cellular factors often reported to befalse positives in two-hybrid experiments. Other factorsfound in our two-hybrid analysis and not discussed indetail include other mitochondrial proteins such asMRPL32, NDUFS2, and ALDH2.

In conclusion, we were able to enlarge the list ofBBS protein binding partners presented by other researchgroups [Ansley et al., 2003; Kim et al., 2004; Badanoet al., 2006]. Furthermore, the known BBS4 interactingfactors PCM1 and DCTN1 have been shown to bind alsoto BBS1 and BBS2. Colocalization studies performedwith a selection of most-promising novel interacting part-ners in HeLa cells (ALDOB, EXOC7, KRT18, FLOT1,PAX2) strongly supported our model of direct interactionsbetween BBS proteins and those factors. Elucidating themolecular basis of the observed interactions, at the sametime analyzing if other BBS proteins, e.g. BBS10, bind tothe same factors, will help to untangle the complex inter-play displayed in Bardet-Biedl syndrome.

ACKNOWLEDGMENT

We are indebted to Mrs. U. Neidel for her experttechnical assistance.

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Novel interaction partners of Bardet-Biedl syndrome proteins