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Originally published In Press as doi:10.1074/jbc.M505062200 on June 13, 2005

J. Biol. Chem., Vol. 280, Issue 30, 27688-27696, July 29, 2005
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Characterization of the Intraflagellar Transport Complex B Core

DIRECT INTERACTION OF THE IFT81 AND IFT74/72 SUBUNITS*{boxs}

Ben F. Lucker{ddagger}, Robert H. Behal{ddagger}, Hongmin Qin§, Laura C. Siron{ddagger}, W. David Taggart{ddagger}, Joel L. Rosenbaum§, and Douglas G. Cole{ddagger}

From the {ddagger}Department of Microbiology, Molecular Biology, and Biochemistry and the Center for Reproductive Biology, LSS142, University of Idaho, Moscow, Idaho 83844 and the §Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520

Received for publication, May 9, 2005 , and in revised form, June 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic cilia and flagella are specialized organelles found at the periphery of cells of diverse organisms. These cellular appendages have been adapted for multiple uses such as bulk fluid movement, cellular motility, and sensing extracellular signals (1–3). The significance of these organelles in human health is becoming increasingly obvious as the list of diseases associated with cilia and flagella continues to expand. These include polycystic kidney disease (4–6), ocular degeneration (7), immotile cilia and Kartagener's syndromes (8, 9), and Bardet-Beidl syndrome (10, 11). Although the functions of cilia and flagella have evolved considerably, all of these organelles appear to be assembled via the same ancient process known as intraflagellar transport or IFT1 (reviewed in Refs. 12 and 13).

IFT is an intracellular, bidirectional movement of protein particles along the length of cilia and flagella (14, 15). Anterograde transport of particles to the distal tip of the organelle is driven by kinesin-2 (15–17), whereas retrograde transport of particles to the cell body is driven by cytoplasmic dynein-1b/2 (18, 19). The IFT particles are composed of at least 17 proteins that form two distinct complexes known as A and B (17, 20). Complex A contains six subunits ranging from 43 to 144 kDa, whereas complex B contains at least 11 subunits ranging from 20 to 172 kDa (12). The IFT motors and particle proteins are well conserved in ciliated organisms (13). In Chlamydomonas reinhardtii, defects in the anterograde motor, kinesin-2, result in reduced delivery of axonemal building blocks and either shortened or absent flagella (15, 16, 21, 22). Defects in the retrograde motor, dynein 1b, result in shortened flagella filled with IFT particles (18, 19, 23, 24). Mutations that affect specific Chlamydomonas IFT particle proteins also result in flagellar assembly phenotypes. For example, deletion of the complex B genes, IFT88 (4) and IFT52 (25, 26), result in flagellaless cells, whereas a temperature-sensitive mutant in IFT172, fla11ts, is defective in both flagellar assembly and remodeling IFT particles at the flagellar tip (27). As in Chlamydomonas, IFT mutants in other ciliated organisms are also characterized by defects in ciliary assembly (13).

Whereas functional studies of IFT have progressed rapidly, little is known about the structure of the IFT particles and complexes. Sequence analysis does reveal that many of the IFT particle proteins contain sequence motifs that are predicted to form protein-protein binding domains (12). These domains include tetratricopeptide repeats (28), WD-40 repeats (29), coiled-coil domains (30), and novel degenerate repeats termed WAA repeats found only in IFT particle proteins (27). Some of these domains, such as tetratricopeptide and WD repeats, are well known for mediating transient protein-protein interactions, whereas coiled-coil domains typically mediate more stable or long term interactions. Transient protein-protein interactions would probably be useful in the binding, transport, and release of specific IFT cargoes, such as radial spoke complexes (31). Transient interactions might also play a role in the assembly, disassembly, and rearrangement of the large IFT particles that occur at the base and tip of the organelle (32). More stable interactions such as those generated by coiled-coils could serve to keep the smaller units such as complexes A and B intact.

Here we report on the cloning of the Chlamydomonas IFT81 gene, which encodes a protein predicted to form extensive coiled-coils (33). IFT81 is shown to be an integral component of a salt-stable fraction of complex B, which also contains IFT88, IFT74, IFT72, IFT52, IFT46, and IFT27 and is now termed the complex B core. IFT74 and IFT72 are both derived from the same gene and are often referred to as IFT74/72 (31). With increased ionic strength, IFT172, IFT80, IFT57, and IFT20 completely dissociate from complex B and appear to be independent of each other. To investigate potential protein-protein interactions within the complex B core, a combination of chemical cross-linking, immunoprecipitation, and MALDI-TOF mass spectrometry was utilized. With this approach, we identified cross-linked products that contained only IFT81 and IFT74/72. Subsequent yeast-based two-hybrid analysis suggested that IFT81 interacts directly with IFT74/72 through a predicted coiled-coil domain. IFT81 was also found to homodimerize in the yeast two-hybrid system. Last, yeast-based three-hybrid analysis supports the hypothesis that IFT81 and IFT74/72 form a higher order oligomer that is consistent with the tetrameric complex (IFT81)2(IFT74/72)2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Reagents—C. reinhardtii strains CC-124, CC-125, and fla2ts (CC-1390) were obtained from the Chlamydomonas Center (available on the World Wide Web at www.chlamy.org/) and maintained on solid TAP medium (34) under continuous light. To harvest flagella, cells were grown in liquid TAP medium under a 16:8 light/dark cycle, bubbling continuously with air.

Cloning of IFT81—A Chlamydomonas cDNA expression library (generously provided by P. Lefebvre) was screened using a set of four anti-IFT81 monoclonal antibodies, 81.1–81.4 (17), as directed by the manufacturer (Stratagene). One clone, p10C, cross-reacted with three of the four antibodies (all but 81.2) and was found to contain the 3'-end 1018 nucleotides of the IFT81 cDNA. The initial and largest open reading frame of p10C encoded the carboxyl-terminal 129 amino acids (14.0 kDa) of the IFT81 protein. In a separate experiment, sucrose density gradient-purified IFT proteins were separated by two-dimensional gel electrophoresis as described previously (17). The IFT81 protein band was excised from the two-dimensional gel, digested with trypsin, and fractionated by reverse phase high pressure liquid chromatography. Edman degradation of two isolated tryptic peptides yielded the following sequences: tr-16, YHMLHCQLHITDQNIK; tr-18, NAEGGGSGAVFSEE. The former peptide was encoded by p10C, whereas the latter was used as a template to design degenerate PCR primers for amplification of additional IFT81 cDNA sequence. This approach allowed us to identify an additional 516 bp of cDNA sequence. In order to complete the sequencing of the IFT81 cDNA, additional cDNA clones were obtained by screening the cDNA library. The IFT81 cDNA sequence was later verified with a full-length cDNA clone (accession number AV630357 [GenBank] ) (35, 36). In order to sequence the IFT81 gene, p10C was used to probe a Chlamydomonas genomic BAC library (Clemson University Genomics Institute, Clemson, SC) (available on the World Wide Web at www.genome.clemson.edu/). Three BAC clones containing the IFT81 gene were verified by PCR, and one was chosen for sequencing to identify the intron/exon structure. Sequencing primers are available upon request.

Northern Analysis—CC-124 gametes were deflagellated by pH shock, and total RNA was isolated and stored as described previously (27). For electrophoresis, total RNA was fractionated on 1.2% formaldehyde agarose gels and then transferred to nylon membranes (Hybond-N; Amersham Biosciences) and probed with 32P-labeled probes according to Ref. 37. Ribosomal RNA stained with ethidium bromide was used as the loading control.

Flagellar Isolation—Flagella were isolated from Chlamydomonas by pH shock as described by Witman et al. (38) with minor modifications as follows. Cells (16–32 liters) were harvested using a Pellicon tangential flow cell concentrator (Millipore Corp., Bedford, MA) to a volume of 1–2 liters. Cells were then collected by centrifugation at 900 x g for 2 min at 25 °C. Cell pellets were resuspended into 4–6 liters of fresh M1 medium (39) and placed under light with continuous air bubbling until nearly all cells were flagellated. Flagellated cells were concentrated as described above, and the cell pellets were resuspended in 500 ml of 10 mM HEPES, pH 7.2; 50% sucrose was added to a final concentration of 5% 10 min prior to deflagellation. The addition of 5% sucrose increased the yield of IFT proteins present in the excised flagella.

To initiate deflagellation, 0.50 M acetic acid was added to lower the pH of vigorously stirred cells to 4.6. After 30 s, deflagellation was confirmed using phase-contrast microscopy, and the suspension was quickly neutralized to a pH of 7.2 using 0.50 M KOH; the cell suspension was placed immediately on ice. All subsequent steps were performed at 4 °C or on ice. Protease inhibitors were added to the following final concentrations: 0.1 mM phenylmethylsulfonyl fluoride, 1.7 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 µg/ml pepstatin A, and 5.0 µg/ml soybean trypsin inhibitor. After 10 min of cooling, most of the cell bodies were removed by centrifugation at 900 x g for 2 min at 4 °C. The supernatant was layered over 13 ml of 30% sucrose cushions in 50-ml conical tubes and centrifuged at 800 x g for 10 min at 4 °C in a swinging bucket rotor. Flagellar supernatants were pooled and further centrifuged at 10,000 rpm for 15 min in a Sorvall SS-34 rotor. Flagellar pellets were resuspended in HMEK buffer (10 mM HEPES, 5 mM MgSO4, 0.5 mM EDTA, 25 mM KCl, pH 7.2) with protease inhibitors present and then centrifuged in a microcentrifuge at 16,100 x g for 10 min. The supernatant was discarded, and flagellar pellets were stored indefinitely at –80 °C.

Preparation and Sizing of the Complex B Core—Flagellar matrix samples were prepared by resuspension of frozen flagellar pellets in HMEK buffer + 300 mM NaCl (HMEK-300) with protease inhibitors. Flagella were mechanically sheared by pipetting at least 80 times with a 200-µl pipettor. Axonemal and insoluble proteins were removed by centrifugation at 16,100 x g for 10 min at 4 °C; the supernatant was considered to be the flagellar matrix consisting primarily of soluble flagellar proteins. Sucrose density gradients (10–25%) were made in 14 x 89-mm tubes with solutions containing HMEK-300. The flagellar matrix was centrifuged through the gradients for 16 h at 37,000 rpm using an SW41-Ti rotor (Beckman) at 5 °C. Gradient fractions (~525 µl) were collected from the bottom using capillary tubing and a peristaltic pump with a flow rate of 1.05 ml/min. Although complex B partially dissociated under these gradient conditions, a core group of complex B subunits including IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, and IFT27, remained together with a sedimentation value of 11.0 S. Sedimentation standards used were as follows: thyroglobulin, 19.3 S, catalase, 11.3 S, bovine serum albumin, 4.65 S, ovalbumin, 3.5 S, and bovine heart cytochrome c, 1.86 S.

In order to determine the diffusion coefficient for the complex B core, the ~11 S sucrose gradient fractions were pooled and loaded onto an FPLC 1.6 x 60-cm Sephacryl S-300 (Amersham Biosciences) gel filtration column equilibrated in HMEK-300. This was repeated with three separate preparations of 11 S complex B core. The complex B core proteins co-eluted at an average peak volume of 40.5 ml, corresponding to a diffusion coefficient of 1.80 x 10–7 cm2 s–1. Gel filtration standards used included thyroglobulin (2.6 x 10–7 cm2 s–1), apoferrition (3.24 x 10–7 cm2 s–1), alcohol dehydrogenase (4.76 x 10–7 cm2 s–1), and bovine serum albumin (6.3 x 10–7 cm2 s–1). The column bed and void volumes were determined with blue dextran and ATP, respectively. The apparent molecular mass of the complex B core were determined with the Svedberg equation (40),

(Eq. 1)
where R is the gas constant, T is temperature in Kelvin, {nu} is the partial specific volume of the protein, {rho} is the solution density, and S and D are the experimentally derived sedimentation and diffusion coefficients, respectively. The partial specific volume was assumed to be 0.72 cm3/g, and the solution density was assumed to be 1.00 g/cm3.

Preparation of Anti-IFT81 Resin—Three monoclonal antibodies raised against IFT81, 81.1, 81.3, and 81.4 (17), were partially purified from mouse ascites fluid using CM-Affi-Gel Blue (Bio-Rad) following the recommended procedures. Antibodies were further purified using protein G-conjugated Sepharose beads (Sigma) as described by Harlow and Lane (41). Protein G-purified antibodies were concentrated to ~10 mg/ml using Centriprep-30 spin columns (Amicon) preincubated with 5% Tween 20 in phosphate-buffered saline and rinsed thoroughly with deionized water just prior to use. Concentrated antibodies were dialyzed against conjugation buffer containing 0.1 M NaHCO3, pH 8.4, 0.5 M NaCl and then conjugated to CNBr-activated Sepharose 4B (Sigma) overnight at 4 °C. The anti-IFT81 resin was then blocked using 1.0 M ethanolamine, pH 8.0, for 2 h at room temperature.

Chemical Cross-linking—Sucrose gradient fractions enriched in the complex B core were pooled and divided into equal aliquots. Gradient fractions were treated with the chemical cross-linkers 1,5-difluoro-2,4-dinitrobenzene (DFDNB; Pierce) or dimethyl adipimidate (DMA; Pierce) at final concentrations of 0.0, 0.03, 0.1, 0.3, and 1.0 mM for 10 min on ice before being quenched with 10 mM Tris-HCl, pH 8.5. The complex B core was then bound to anti-IFT81 antibody resin. The supernatant was removed, and the resin was washed three times with 15 bed volumes of HMEK-300. The proteins were eluted from the resin by boiling in an equal volume of 2x SDS sample buffer. Immunoprecipitates were separated on 4.0, 5.0, 6.0, or 7.5% SDS-polyacrylamide gels (42) and visualized with Coomassie Blue. Protein bands containing cross-linked products were excised from the gels, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Resulting peptide masses were compared with expected IFT81 and IFT74/72 peptides using Protein Prospector (43).

Yeast-based Two- and Three-hybrid Analysis—Hybrizap 2.1 yeast two-hybrid vectors and the YRG-2 yeast strain were obtained from Stratagene, whereas the yeast three-hybrid pBridge vector was obtained from BD Biosciences. All IFT81 constructs for this analysis were PCR-amplified and subcloned using a full-length cDNA clone, LCL077g05_r (Accession number AV630357 [GenBank] ; Kazusa DNA Research Institute, Kisarazu, Chiba, Japan) (35, 36), as a template. The IFT81 constructs used for two-hybrid and three-hybrid analyses include 81F (amino acids 1–683), 81CC12 (amino acids 125–683), 81N (amino acids 1–128), 81CC1 (amino acids 125–454), 81CC2 (amino acids 446–632), and 81C (amino acids 625–683). In brief, all PCR products of IFT81 were first ligated into TOPO Zero-Blunt pcr4 vectors according to the manufacturer (Invitrogen). Each cDNA insert was excised by restriction digest and ligated into the two-hybrid vectors, pAD-GAL4–2.1 and pBD-GAL4-Cam, using T4 DNA ligase (Invitrogen) according to standard procedures.

The cloning of the IFT74/72-Mid construct containing amino acids 210–397 was previously described (31). The other IFT74/72 constructs were generated from a full-length IFT74/72 cDNA template, which was generated as follows. Total Chlamydomonas RNA was isolated from strain CC-124 30 min after deflagellation as previously described (27). First strand cDNA synthesis was performed using the ThermoScript reverse transcription-PCR system (Invitrogen). Second strand synthesis was carried out using the following IFT74/72 gene-specific primers: upper primer, 5'-ATGGACAGGCCCTCTAGCCGCG-3'; lower primer, 5'-TACACCACGTTCTTGAC-3'. The resulting full-length IFT74/72 cDNA was cloned into the TOPO Zero-Blunt pcr4 vector and used as a template to PCR-amplify IFT74/72 cDNA to prepare the AD and BD two-hybrid constructs as described above for IFT81. The resulting IFT74/72 constructs are 74F (amino acids 1–641), 74N (amino acids 1–137), 74CC1 (amino acids 137–457), 74NCC1 (amino acids 1–457), 74MID (amino acids 210–610), and 74CC2 (amino acids 424–563).

Three-hybrid analysis utilized the pBridge vector (BD Biosciences), which contains two multiple cloning sites, MCS-1 and MCS-2. MCS-1 generates a DNA-binding domain (BD) fusion protein under the regulation of the alcohol dehydrogenase promoter, whereas MCS-2 is responsible for expression of a second protein under the regulation of the MET25 promoter. The full-length 74F was ligated into the pBridge MCS-1 using the EcoRI and SalI restriction sites, whereas 81F was ligated into MCS-2 using the NotI and BglII restriction sites producing the pBri-74F-81F plasmid. As a control, 74F was ligated into MCS-1 without placing any inserts into MCS-2 producing the plasmid, pBri-74F.

All yeast media were made according to the Hybrizap 2.1 manual (Stratagene). Yeast transformations were essentially performed as described by Gietz and Woods (44). Briefly, yeast were grown on solid YAPD (20 g/liter Difco peptone, 10 g/liter yeast extract, 20 g/liter agar, 40 mg/liter adenine sulfate, 2% (v/v) glucose, pH 5.8) medium for 2–3 days. Cells (25 µl/transformation) were transferred to 1.0 ml of sterile water and centrifuged for 5 s at 16,000 x g. Cell pellets were resuspended with 1.0 ml of sterile 100 mM LiOAc at 30 °C and incubated for 5 min at 30 °C. Cells were split into equal aliquots representing the number of transformations and centrifuged at 16,000 x g for 5 s. The supernatant was removed, and the following reagents were added to the cell pellet in order, 240 µl of polyethylene glycol 3350 (50% w/v, filter-sterilized), 36 µl of 1.0 M LiOAc, 60 µl of sheared salmon sperm (2.0 mg/ml, boiled 5 min, and set on ice for at least 30–60 s), 3 µl of plasmid DNA (0.2–1 µg/µl). Each sample was vortexed vigorously for 3 min and then incubated for 20 min at 42 °C. Cells were centrifuged at 16,000 x g for 10 s and then gently resuspended in 200 µl of sterile water with wide bore pipette tips and plated onto appropriate dropout media. Yeast whole cell lysates were prepared using the yeast protein extraction reagent, YPER (Pierce), and analyzed by Western blotting to confirm fusion protein expression. Anti-Gal4-AD and anti-Gal4-BD monoclonal antibodies were purchased from BD Biosciences.



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  		FIG. 1.
IFT complex B partially dissociates with increased ionic strength. Flagellar matrix was fractionated through a 13-ml 10–25% sucrose density gradient. A, silver-stained 7.5% acrylamide gel. B, Western blots probed with anti-IFT antibodies as indicated. Complex A proteins (IFT144 and -139) co-sediment at 16 S, whereas complex B dissociates into different components. The complex B core proteins co-sediment at 11 S and include IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, and IFT27. IFT172, IFT80, IFT57, and IFT20 fully dissociate from complex B under these conditions.

 
The two-hybrid strong interaction control consisted of amino acids 132–236 of wild-type {lambda} cI, fragment C, fused to both the AD and BD proteins. This protein is able to homodimerize with a Kd of 20 nM (Stratagene). The weak interaction control consisted of a mutant form of the {lambda} cI protein fused to both the AD and BD proteins. The mutant form of the protein has a homodimerization Kd of 200 nM. It should be noted, however, that Estojak et al. (45) determined that interactions identified in yeast two-hybrid analysis do not have a linear correlation to Kd values determined with in vitro binding assays. Thus, identification of two-hybrid interactions that are similar to either the strong positive or weak positive control does not allow us to estimate Kd values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Partial Dissociation of IFT Complex B Reveals a Stable Core—Under low salt conditions, Chlamydomonas IFT proteins are known to separate into two complexes known as A and B (17). By increasing the ionic strength, complex B, but not A, is disrupted. In this study, when flagellar extract (matrix) was fractionated on sucrose density gradients containing the low salt buffer, HMEK, supplemented with 300 mM NaCl (HMEK-300), IFT complex B dissociated into several components, whereas complex A remained intact at ~16 S (Fig. 1). The dissociated B subunits included IFT20, which was found near the top of the gradient at ~3.5 S and IFT57, which peaked at ~7.5 S. The additional dissociated subunits included IFT172 and IFT80 (CHE2), which co-sedimented at ~6.5 S. Hydrodynamic measurements (27) and immunoprecipitations of IFT172 (data not shown), however, lead us to conclude that IFT172 and IFT80 are not associated with one another under these conditions. In contrast, IFT81, IFT74, IFT72, IFT52, IFT46, and IFT27 co-sedimented as a single core complex at 11.0 S, whereas IFT88 was spread out over several fractions, suggesting that only a portion of IFT88 was associated with the complex B core under these conditions.

The core subunits that co-sedimented at 11 S in HMEK-300 were also found to co-elute together from a Sephacryl S-300 gel filtration column (Fig. 2). Based on this elution, the diffusion coefficient of the complex B core was determined to be 1.80 x 10–7 cm2 s–1. Combining the sedimentation value and diffusion coefficient in the Svedberg equation (40), the approximate molecular mass of the complex B core was calculated to be 5.0 x 105 Da. Based on predicted protein masses, the total mass of a complex containing one copy each of IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, and IFT27 is predicted to be 4.2 x 105 Da. If the mass of IFT88, which appears to be present at substoichiometric levels, is omitted, the predicted mass is only 3.3 x 105 Da. Densitometry of the complex B core reveals that IFT81 stains with Coomassie Blue at a level that is 1.4-fold higher than either IFT74 or IFT72 (data not shown), suggesting that IFT 81 may be present at a 2-fold stoichiometry relative to the other complex B subunits. If two copies of IFT81 were present in each complex B core, the summed mass of the core would be predicted to be either 4.1 x 105 Da in the absence of IFT88 or 4.9 x 105 Da when IFT88 is bound. Thus, the experimentally calculated molecular mass of 5.0 x 105 Da for the complex B core is more consistent with two copies of IFT81. Since this approach, however, is not precise, we cannot rule out the possibility that only one IFT81 subunit is present in each IFT complex B.



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  		FIG. 2.
The IFT complex B core subunits co-elute from S-300 gel filtration. Following fractionation on a 10–25% sucrose density gradient in HMEK-300, the complex B core was applied to a Sephacryl S-300 16/60 FPLC column equilibrated in HMEK-300. 2.0-ml fractions were collected, fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies as indicated. The arrows, from left to right, indicate elution position of calibration standards, thyroglobulin (T), apoferritin (A), alcohol dehydrogenase (ADH), bovine serum albumin (BSA), and ovalbumin (OV), respectively.

 
IFT81 Is a Coiled-coil Protein—Anti-IFT81 monoclonal antibody (17) screening of a Chlamydomonas cDNA expression library was used to identify a partial IFT81 cDNA, which served as a probe to identify additional cDNA clones and to screen for Chlamydomonas genomic BAC clones. cDNA clones were used to elucidate the complete IFT81 cDNA, whereas the BAC clones were used to identify the intron-exon structure of the IFT81 gene. The IFT81 gene contains 13 exons interrupted by 12 introns as shown in Fig. 3A. Shotgun cloning of one IFT81 BAC clone identified a number of Chlamydomonas genes, including S926, a nuclear marker for linkage group XIX (data not shown). According to the Department of Energy Joint Genome Institute Chlamydomonas genome project, the IFT81 gene falls on scaffold 1 (genome version 2.0; available on the World Wide Web at genome.jgipsf.org/chlre2/chlre2.home.html) and is within 19,000 bp of the S926 gene (46).

The Chlamydomonas IFT81 cDNA has an open reading frame that encodes a 683-amino acid protein with a predicted molecular mass of 77,073 Da and pI of 6.25. Both values are close to the experimentally derived mass of 81 kDa and pI of 6.1 (17). IFT81 is similar to two previously identified complex B subunits, IFT74 and IFT72 (31). Since IFT74 and IFT72 are nearly identical and are encoded by the same gene, they are often referred to as IFT74/72 (31). Although IFT81 and IFT74/72 are only weakly related at the amino acid level (13.4% identity; 43.5% similarity), they share similar domain structure with respect to predicted formation of coiled-coil domains (Fig. 3B) (33). Like other flagellar genes, expression of both IFT81 and IFT74/72 is up-regulated within 15 min following deflagellation (Fig. 3C). As observed with IFT74/72 (31), the IFT81 protein has been conserved in ciliated organisms (supplemental Fig. S1), homologues to IFT74/72 and IFT81 have not been identified in nonciliated organisms.

Chemical Cross-linking of IFT81 and IFT74/72—In order to better understand the architecture of the IFT complex B core, we initiated a series of chemical cross-linking experiments. The 11 S complex B core fractions were pooled from sucrose density gradients and treated with one of two cross-linking reagents, DFDNB or DMA. After cross-linking was quenched, the complex B core was precipitated with anti-IFT81 resin followed by separation on low percentage acrylamide SDS-PAGE (Fig. 4). Immunoprecipitation generated a 10–20-fold concentration of the proteins of interest concomitant with removal of background proteins. With increasing concentration of cross-linker, high mobility protein bands corresponding to unique cross-linked species appeared on the gels. A DFDNB cross-linked product with a relative mobility of ~200 kDa (Fig. 4A, asterisk) was excised, digested with trypsin, and analyzed using MALDI-TOF mass spectrometry. This analysis yielded 25 peptide masses that ranged in size from 700 to 2300 Da. 13 of the masses matched theoretical tryptic peptides from IFT81, whereas 11 masses matched IFT74/72 tryptic peptides (Table I). One mass did not match any predicted IFT tryptic peptide. Thus, we conclude that only IFT81 and IFT74/72 were present in that specific cross-linked sample.


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  		TABLE I
Tryptic peptides from DFDNB cross-linked IFT81 and IFT74/72

Tryptic digestion produced 25 masses that were identified in the range of 630-2300 Da. Thirteen matched predicted IFT81 tryptic peptides, whereas 11 matched predicted IFT74 tryptic peptides. Several peptides contained missed tryptic cleavages, and three contained an oxidized methionine (Met-ox). Only one mass (1428.6700 Da) could not be attributed to any IFT protein.

 



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  		FIG. 3.
IFT81 is a coiled-coil protein. A, the IFT81 gene contains 13 exons with 12 introns (base pair sizes are shown). The IFT81 gene produces a cDNA with an open reading frame of 2,052 bp including the termination codon. B, the IFT81 gene encodes a 683-amino acid protein with two regions predicted to form primarily coiled-coil secondary structure. C, IFT81 and IFT74/72 mRNA both increase in response to deflagellation. Samples include nondeflagellated (NDF) and 15 and 60 min postdeflagellation. TUB, {beta}-tubulin mRNA. Total RNA is visualized with ethidium bromide.

 
The rate at which cross-linked proteins migrated through the SDS-PAGE gels was sensitive to the amount of acrylamide used to form each gel. For example, experiments using DMA yielded a cross-linked IFT81-IFT74/72 product with a relative mobility of ~250 kDa when separated by SDS-PAGE using 7.5% acrylamide (Fig. 4B). When fractionated on a 4% gel, however, the same cross-linked species was resolved into two distinct protein bands with relative mobilities of ~165 and ~158 kDa (Fig. 4C). Subsequent mass spectrometric analyses of these two samples revealed that both contained only IFT81 and IFT74/72. We conclude that these two separate species represented distinct cross-linking events between IFT81 and IFT74/72. It is possible that the slower mobility species was a product of IFT81-IFT74, whereas the faster mobility species was a product of IFT81-IFT72.

IFT81 Can Interact with the First Coiled-coil Domain of IFT74/72—A GAL4-based yeast two-hybrid system was used to test the hypothesis that IFT81 and IFT74/72 interact directly (47). Full-length open reading frame cDNA for IFT81 (81F) and IFT74/72 (74F) were separately cloned into both the binding domain (pBD) and activation domain (pAD) vectors. As demonstrated in Fig. 5 (lanes 5 and 6), strains that were co-transformed with either AD-81F/BD-74F or AD-74F/BD-81F allowed the yeast to grow on selective defined medium lacking histidine. Comparison of serial dilutions of these strains relative to positive interaction controls demonstrates that IFT81 and IFT74 interact strongly in this assay.

To determine which portions of IFT81 and IFT74/72 were required for interaction, deletion constructs were generated for both the AD and BD vectors and assayed for interactions. Serial dilutions of transformed yeast strains were plated on selective media as shown in Fig. 5; colony growth in the absence of histidine is recorded in Fig. 6. In this assay, all IFT74/72 constructs that expressed the first predicted coiled-coil domain showed a strong interaction with the full-length IFT81 protein (Fig. 6A). Indeed, this domain (CC1; residues 125–454) by itself was sufficient to produce an interaction with IFT81. In contrast to IFT74/72, IFT81 constructs required the inclusion of both coiled-coil domains in order to interact with full-length IFT74/72 (Fig. 6B). Unexpectedly, IFT81 also displayed a weak interaction with itself (Fig. 6C). This raised the possibility that IFT81 might homodimerize in IFT complex B and suggested that IFT81 and IFT74/72 are capable of forming a higher order oligomer. It should be noted that the same two IFT81 constructs that were able to homodimerize were also the only two constructs capable of interacting with full-length IFT74/72. In contrast to IFT81, IFT74/72 was not capable of interacting with itself in the yeast two-hybrid system.



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  		FIG. 4.
Chemical cross-linking of IFT81 and IFT74/72. A, sucrose density gradient-purified complex B core was treated with increasing concentrations of DFDNB and subsequently precipitated using anti-IFT81 resin and separated by SDS-PAGE using 6% acrylamide. The first lane contains molecular mass standards. A DFDNB-cross-linked product with relative mobility of 200 kDa (*) was excised, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry; IFT81 and IFT74/72 were the only IFT proteins present in this band. B, 1.0 mM DMA cross-linking of the complex B core yields an IFT81-IFT74/72 cross-linked product (*) with relative mobility of ~250 kDa when separated by SDS-PAGE using 7.5% acrylamide. C, the relative mobility of the DMA-IFT81-IFT74/72 product shown in B was reduced, and the product was resolved into two protein bands with relative mobilities of 158 and 165 kDa when fractionated on a 4% acrylamide gel. These two protein bands contained only IFT81 and IFT74/72 and probably represent separate cross-linking sites, each affecting the relative mobility accordingly. All protein gels contained a ratio of 37.5:1 acrylamide/bisacrylamide and were stained with Coomassie Blue following electrophoresis.

 
IFT81 and IFT74/72 Can Form a Higher Order Complex—A GAL4-based yeast three-hybrid system was used to test the hypothesis that IFT81 and IFT74/72 can form a higher order oligomer. This particular three-hybrid assay differs from the GAL4-based two-hybrid assay by controlling the expression of a third protein that might enhance or interfere with the interaction between two proteins (48). Whereas the AD vector can be identical to those used in the two-hybrid assays discussed above, the pBridge vector contains two multiple cloning sites. The first site, MCS-1, generates a BD fusion protein under the regulation of the alcohol dehydrogenase promoter. The second site, MCS-2, which is responsible for expression of a third protein, is under the regulation of the MET25 promoter. Thus, in the absence of methionine, a third protein is expressed, and its effect on enhancing or inhibiting the interaction between the AD and BD fusion proteins can be analyzed. In practice, however, we found that the MET25 promoter is leaky in the presence of methionine. When 81F was placed into MCS-2, HA-tagged full-length IFT81 protein was expressed in yeast grown with or without methionine in the medium (Fig. 7A). To circumvent this problem, a control vector was generated in which no IFT81 cDNA was placed in MCS-2; this eliminated the possibility of IFT81 expression.

To determine whether IFT81 was able to "bridge" an interaction between BD-74F and various AD-74 deletion constructs, yeast strains were transformed with one of two pBridge vectors. The first vector contained both pBD-74F and HA-81F (termed pBri-74F-81F); the second, control vector contained pBD-74F with an empty MCS-2 (termed pBri-74F). Both pBridge vectors were tested against all of the previously described pAD-74 constructs. As a positive control, the pBri-74F vector was tested against pAD-81F, which showed the expected strong interaction (Fig. 7B, lane 1). In the absence of IFT81 expression, no positive interaction was observed between BD-74F and the AD-74 constructs (Fig. 7B, lanes 2–7). When full-length IFT81 was expressed, however, positive interactions were observed between BD-74F and some of the AD-74 constructs (Fig. 7C, lanes 1–7). Interestingly, the AD-74 constructs that showed interaction were identical to the deletion constructs that showed interaction with full-length IFT81 (Fig. 6A). These data indicate that IFT81 is capable of bringing together two or more copies of the IFT74 protein.

The Human Homologues of IFT81 and IFT74/72 Interact— The mammalian homologues of Chlamydomonas IFT81 (CrIFT81) and IFT74/72 (CrIFT74) are known as CDV-1r (49) and CMG-1 (50), respectively. The overall amino acid identity between CrIFT81 and HsCDV-1r is 29% (54% similarity), and identity between CrIFT74 and HsCMG-1 is 24% (50% similarity). Lupas analysis (51) reveals that HsCDV-1r and CMG-1 are expected to form coiled-coil domains similar to CrIFT81 and CrIFT74, (data not shown). Like CrIFT81 and CrIFT74, yeast-based two-hybrid analysis revealed that strong interactions could occur between full-length HsCDV-1r and full-length HsCMG-1 (Table II). In contrast to CrIFT81, however, HsCDV-1r did not show any indication of homodimerization. Last, the human CMG-1 was capable of homodimerization in the two-hybrid assay, but the slow colony growth suggested a weak interaction.


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  		TABLE II
Yeast-based two-hybrid interactions between human CMG-1 and CDV-1R

+++++, strong growth; ++, weak growth; -, no growth observed; ND, not determined.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Complex B Core Is Defined by Seven IFT Proteins— Chlamydomonas IFT particle proteins can be separated into two complexes, A and B, which contain at least 6 and 11 subunits, respectively (17, 52). It was previously reported that the addition of 50 mM NaCl to HMEK buffer was sufficient to cause IFT172 to partially dissociate from complex B (17), whereas the addition of 100 mM NaCl was sufficient to cause IFT172 to fully dissociate from complex B and behave as a monomeric protein (27). We have shown here that complex B can be further disrupted by supplementing HMEK buffer with 300 mM NaCl (Fig. 1). Under these conditions, IFT172, -80, -57, and -20 fully dissociated, and IFT88 partially dissociated from complex B. The 11.0 S complex B core contained IFT88, -81, -74, -72, -52, -46, and -27. Subsequent gel filtration under these same conditions revealed that all of the complex B core subunits stayed together and co-eluted with a relatively small diffusion coefficient of 1.80 x 10–7 cm2/s (Fig. 2). These hydrodynamic measurements were used to predict a core molecular mass of 504 kDa. This mass is consistent with a core that contains two IFT81 subunits and one each of the IFT88, -74, -72, -52, -46, and -27 subunits, which have a total predicted mass of 492 kDa (Fig. 8). The calculations based on hydrodynamic measurements, however, are an estimate. Thus, although gel densitometry indicates that IFT81 is present at a 1.4-fold stoichiometry relative to IFT74 and IFT72, we cannot rule out the possibilities that 1) only a single copy of IFT81 is present in each complex B or 2) that the stoichiometry of the IFT81 subunit may vary from complex to complex. Last, it should be noted that the complex B core subunits do not require the presence of IFT172, -80, -57, or -20 to remain bound to the core.



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  		FIG. 5.
Full-length IFT81 and IFT74/72 interact strongly according to yeast-based two-hybrid analysis. Serial dilutions of yeast strains (1:10 beginning with 105 cells) containing both BD and AD plasmid constructs are grown on nonrestrictive media (–Leu, –Trp; top panels) and restrictive media (–Leu, –Trp, –His; bottom panels). Lanes 1–4, full-length IFT81 and full-length IFT74 AD and BD constructs are tested against control vectors. Lanes 5 and 6, full-length IFT81 and IFT74 AD or BD vectors tested against each other. Lanes 7–9, strong, weak, and negative interacting controls, respectively.

 



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  		FIG. 6.
Yeast-based two-hybrid interactions using deletion constructs of IFT81 and IFT74/72. The number of plus signs indicates the strength of interaction, based on growth of serially diluted cells on solid media. +++++, growth at a dilution that contained only 101 cells; +, growth upon spotting of 105 cells. – – –, no growth occurred when 105 cells were plated. A, full-length AD- or BD-81 was tested against various IFT74 constructs. B, full-length AD- or BD-74 was tested against various IFT81 constructs. C, full-length BD-81 was tested against various IFT81 constructs.

 



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  		FIG. 7.
Yeast-based three-hybrid analysis of IFT81 and IFT74. The ability of IFT81 to mediate an interaction between IFT74 and IFT74 was tested. A, Western blot analysis using anti-IFT81 of whole yeast cells containing the pBridge vector with IFT74 in MCS-1 and IFT81 in MCS-2 (pBri-74F-81F). Asterisks, the full-length HA-IFT81F fusion protein. The IFT81 protein is expressed in both the presence and absence of methionine. The third lane is a control containing cells transformed with the pBridge vector containing IFT74 in MCS-1 with no IFT81 inserted in MCS-2. B and C, dilution series of transformed yeast strains plated on selective media. Media lacking histidine selected for an interaction, whereas media lacking methionine drove expression of inserts at MCS-2. B, lane 1, positive interaction control; a yeast strain expressing both AD-81F and pBri-74F but lacking an insert at MCS-2 of the pBridge vector. Lanes 2–7, yeast strains were co-transformed with both the pBri-74F vector and various AD-74 deletion constructs. No growth is observed in the absence of histidine. C, lanes 1–6, cells were co-transformed with the pBri-74F-81F vector and various AD-74 deletion constructs. Lane 7, control strain co-transformed with AD-MCS (no insert) and pBri-74F-81F.

 
Are Weak Interactions between IFT Proteins Indicative of IFT Particle Rearrangements?—As reported here and previously (17, 20, 27, 52), the large, several hundred-µm-long IFT particles visualized by electron microscopy (15) are easily disrupted to yield soluble 16–17 S complexes (A and B), and one of these complexes (B) can be further disrupted by mild increases in ionic strength. This suggests that the IFT particles and possibly the IFT complexes themselves are held together by relatively weak interactions. It is possible that these weak interactions are indicative of the dynamic nature of IFT. At the base of the flagellum, for example, IFT proteins must arrange themselves into the very large (hundreds of µm) IFT particles that form at the base and then move along the length of the organelle. Furthermore, since retrograde IFT particles appear smaller than and outnumber the anterograde particles, it has been hypothesized that the IFT particles undergo a rearrangement at the distal tip of the organelle (27, 32). It is reasonable to think that some of the weak interactions observed in vitro may be functioning to facilitate in vivo rearrangements.



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  		FIG. 8.
IFT complex B core model. The Chlamydomonas IFT complex B partially dissociates with increased ionic strength (300 mM NaCl) to reveal a core complex containing IFT88, IFT81, IFT74, IFT72, IFT52, IFT46, and IFT27. The results shown here indicate that IFT81 forms a higher order oligomer with IFT74 and IFT72. We propose that this oligomer is a tetramer. The associations of IFT88, IFT52, IFT46, and IFT27 within the core are unknown.

 
Chemically Cross-linked Proteins Can Run Anomalously on Protein Gels—Due to the sensitivity of mass spectrometry, chemical cross-linking of proteins is enjoying renewed interest. Cross-linked species can be separated by one dimensional SDS-PAGE, excised, and proteolytically digested. Subsequent mass spectrometry analysis of the peptides yields a list of masses that can be compared with predicted peptide masses from known proteins in data sets as large as complete genomes. What should be noted, however, is that the relative electrophoretic mobility of a cross-linked pair of proteins appears to be dependent on both the nature of the cross-link and the nature of the acrylamide gel. The standard treatment of proteins with SDS and reducing agents results in linearized proteins with consistent charge/mass ratios that will move through an acrylamide gel in an electric field in a predictable manner. Indeed, proteins are routinely sized by comparing their relative mobility with a set of standard molecular weight marker proteins. Unless two cross-linked proteins, however, are connected end-to-end, the resulting cross-linked species will not behave as a linear polymer. We demonstrate this phenomenon with DMA-cross-linked IFT81 and IFT74/72, which have an apparent relative mobility of 250 kDa when fractionated on a 7.5% acrylamide gel (Fig. 4B). Fractionation on a 4% acrylamide gel, however, revealed two distinct cross-linked species containing both IFT81 and IFT74 and whose relative mobilities decreased to 158 and 165 kDa (Fig. 4C). Since both species contained only IFT81 and IFT74/72, we conclude that each represented a unique cross-linking event.

IFT81 and IFT74/72 Can Form a Higher Order Oligomer— We show here that IFT81 and IFT74/72 co-purify with the complex B core (Figs. 1 and 2) and that they can be chemically cross-linked to one another (Fig. 4). Yeast-based two-hybrid analysis was used to show that these two subunits can physically interact. Indeed, the first coiled-coil domain of IFT74/72 was sufficient to produce an interaction with full-length IFT81. In contrast, IFT81 required both coiled-coil domains to interact with full-length IFT74. Significantly, IFT81 but not IFT74/72 was capable of homodimerization in the two-hybrid assay. Since there appears to be both one IFT74 and one IFT72 in each complex B, we shifted the analysis of these proteins to a yeast-based three-hybrid system, which allows expression of a third protein. IFT74 was expressed as fusions with both the DNA-binding domain (BD-74) and the activation domain (AD-74). As with the two-hybrid assay, no interaction was observed between BD-74 and AD-74 in the absence of IFT81, but when full-length IFT81 was expressed, BD-74 and AD-74 came together in a complex to allow the yeast to grow on selective media. These results indicate that IFT81 and IFT74/72 are capable of forming a higher order oligomer. This analysis, however, does not allow us to distinguish between a trimer containing one copy of IFT81 and two copies of IFT74/72 or a tetramer that also contains a second copy of IFT81. Hydrodynamic measurements of the complex B core favor the tetramer over the trimer, whereas gel densitometry favors neither. Although we cannot rule out the possibility of a trimer, we put forth a working model that IFT complex B contains a tetramer of 2 x IFT81, 1 x IFT74, and 1 x IFT72, which serves as a central scaffold to which the rest of the complex B subunits bind (Fig. 8).

The IFT81-IFT74/72 Interaction Is Conserved—Like the Chlamydomonas proteins, the human homologues of IFT81 and IFT74/72, CDV-1r and CMG-1 were found to interact with one another in the yeast-based two-hybrid assay (Table II). This result was not unexpected, because recent evidence has indicated that the vertebrate IFT81 and IFT74/72 could be found in cilia. For example, a proteomic study of human tracheal cilia identified the presence of both CDV-1r and CMG-1 (53). More recently, CMG-1 has been immunolocalized in the primary cilia of human umbilical vein endothelial cells (54). Consistent with a ciliary and flagellar role are the observations that the CDV-1r and CMG-1 genes are preferentially expressed in ciliated tissues such as the testes, liver, kidney, brain, and lungs (49, 50). Also, corresponding to sperm development, transcription of the CDV-1r gene is dramatically up-regulated in mouse testes beginning at ~30 days after birth (55, 56). Last, mutations affecting the zebrafish IFT81 gene result in cystic kidneys (57), a phenotype commonly observed with vertebrates carrying mutations in IFT genes (5).

Conclusion—IFT complex B is composed of at least 11 different IFT proteins, some of which can be dissociated under relatively mild conditions to reveal a more stable set of proteins termed the complex B core. Three of the proteins found in this core are IFT81, IFT74, and IFT72. We found that IFT81 could be routinely cross-linked to either IFT74 or IFT72; in this study, we were unable to distinguish between these two nearly identical proteins. Combining two-hybrid and three-hybrid analyses, we have shown that IFT81 and IFT74/72 can directly interact to form a higher order oligomer. Based on these data, we propose that each IFT complex B contains a central tetrameric subcomplex containing two subunits of IFT81 and two subunits of IFT74/72.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM14642 (to J. L. R.) and GM61920 and P20RR16454 (to D. G. C.) from the National Center for Research Resources (Biomedical Research Infrastructure Network). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

To whom correspondence should be addressed: Dept. of Microbiology, Molecular Biology, and Biochemistry, Life Science South 142, University of Idaho, Moscow, ID 83844-3052. Tel.: 208-885-4071; Fax: 208-885-6518; E-mail: dcole{at}uidaho.edu.

1 The abbreviations used are: IFT, intraflagellar transport; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; DFDNB, 1,5-diflouro-2,4-dinitrobenzene; DMA, dimethyl adipimidate; BAC, bacterial artificial chromosome; HA, hemagglutinin; BD, DNA-binding domain; AD, DNA activation domain; FPLC, fast protein liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Pete Lefebvre at the University of Minnesota for the generous gift of a Chlamydomonas cDNA library and Samantha Reed and Greta Anderson at the University of Idaho for expert technical assistance.



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 RESULTS
 DISCUSSION
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