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• W2032141866 abstract "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. 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. 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 (1Bray D. Cell Movements: From Molecules to Motility. Garland Publishing, New York, NY2001Google Scholar, 2Wheatley D. Cell Biol. Int. 2004; 28: 75-77Crossref PubMed Scopus (10) Google Scholar, 3Praetorius H.A. Spring K.R. Annu. Rev. Physiol. 2005; 67: 515-529Crossref PubMed Scopus (229) Google Scholar). 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 (4Pazour G.J. Dickert B.L. Vucica Y. Seeley E.S. Rosenbaum J.L. Witman G.B. Cole D.G. J. Cell Biol. 2000; 151: 709-718Crossref PubMed Scopus (863) Google Scholar, 5Pazour G.J. J. Am. Soc. Nephrol. 2004; 15: 2528-2536Crossref PubMed Scopus (155) Google Scholar, 6Wilson P.D. Int. J. Biochem. Cell Biol. 2004; 36: 1868-1873Crossref PubMed Scopus (106) Google Scholar), ocular degeneration (7Besharse J.C. Baker S.A. Luby-Phelps K. Pazour G.J. Adv. Exp. Med. Biol. 2003; 533: 157-164Crossref PubMed Scopus (40) Google Scholar), immotile cilia and Kartagener's syndromes (8Geremek M. Witt M. J. Appl. Genet. 2004; 45: 347-361PubMed Google Scholar, 9Nonaka S. Tanaka Y. Okada Y. Takeda S. Harada A. Kanai Y. Kido M. Hirokawa N. Cell. 1998; 95: 829-837Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar), and Bardet-Beidl syndrome (10Li J.B. Gerdes J.M. Haycraft C.J. Fan Y. Teslovich T.M. May-Simera H. Li H. Blacque O.E. Li L. Leitch C.C. Lewis R.A. Green J.S. Parfrey P.S. Leroux M.R. Davidson W.S. Beales P.L. Guay-Woodford L.M. Yoder B.K. Stormo G.D. Katsanis N. Dutcher S.K. Cell. 2004; 117: 541-552Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 11Blacque O.E. Reardon M.J. Chunmei L. McCarthy J. Mahjoub M.R. Ansley S.J. Badano J.L. Mah A.K. Beales P.L. Davidson W.S. Johnsen R.C. Audeh M. Plasterk R.H.A. Baillie D.L. Katsanis N.K. Quarmby L.M. Wicks S.R. Leroux M.R. Genes Dev. 2004; 18: 1630-1642Crossref PubMed Scopus (286) Google Scholar). 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 IFT 1The 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. (reviewed in Refs. 12Cole D.G. Traffic. 2003; 4: 435-442Crossref PubMed Scopus (195) Google Scholar and 13Scholey J.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 423-443Crossref PubMed Scopus (337) Google Scholar). IFT is an intracellular, bidirectional movement of protein particles along the length of cilia and flagella (14Kozminski K.G. Johnson K.A. Forscher P. Rosenbaum J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5519-5523Crossref PubMed Scopus (727) Google Scholar, 15Kozminski K.G. Beech P.L. Rosenbaum J.L. J. Cell Biol. 1995; 131: 1517-1527Crossref PubMed Scopus (444) Google Scholar). Anterograde transport of particles to the distal tip of the organelle is driven by kinesin-2 (15Kozminski K.G. Beech P.L. Rosenbaum J.L. J. Cell Biol. 1995; 131: 1517-1527Crossref PubMed Scopus (444) Google Scholar, 16Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar, 17Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar), whereas retrograde transport of particles to the cell body is driven by cytoplasmic dynein-1b/2 (18Pazour G.J. Dickert B.L. Witman G.B. J. Cell Biol. 1999; 144: 473-481Crossref PubMed Scopus (366) Google Scholar, 19Porter M.E. Bower R. Knott J.A. Byrd P. Dentler W. Mol. Biol. Cell. 1999; 10: 693-712Crossref PubMed Scopus (274) Google Scholar). The IFT particles are composed of at least 17 proteins that form two distinct complexes known as A and B (17Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar, 20Piperno G. Mead K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4457-4462Crossref PubMed Scopus (224) Google Scholar). 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 (12Cole D.G. Traffic. 2003; 4: 435-442Crossref PubMed Scopus (195) Google Scholar). The IFT motors and particle proteins are well conserved in ciliated organisms (13Scholey J.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 423-443Crossref PubMed Scopus (337) Google Scholar). In Chlamydomonas reinhardtii, defects in the anterograde motor, kinesin-2, result in reduced delivery of axonemal building blocks and either shortened or absent flagella (15Kozminski K.G. Beech P.L. Rosenbaum J.L. J. Cell Biol. 1995; 131: 1517-1527Crossref PubMed Scopus (444) Google Scholar, 16Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar, 21Mueller J. Perrone C.A. Bower R. Cole D.G. Porter M.E. Mol. Biol. Cell. 2005; 16: 1341-1354Crossref PubMed Scopus (85) Google Scholar, 22Matsuura K. Lefebvre P.A. Kamiya R. Hirono M. Cell Motil. Cytoskeleton. 2002; 52: 195-201Crossref PubMed Scopus (35) Google Scholar). Defects in the retrograde motor, dynein 1b, result in shortened flagella filled with IFT particles (18Pazour G.J. Dickert B.L. Witman G.B. J. Cell Biol. 1999; 144: 473-481Crossref PubMed Scopus (366) Google Scholar, 19Porter M.E. Bower R. Knott J.A. Byrd P. Dentler W. Mol. Biol. Cell. 1999; 10: 693-712Crossref PubMed Scopus (274) Google Scholar, 23Perrone C.A. Tritschler D. Taulman P. Bower R. Yoder B.K. Porter M.E. Mol. Biol. Cell. 2003; 14: 2041-2056Crossref PubMed Scopus (107) Google Scholar, 24Hou Y. Pazour G.J. Witman G.B. Mol. Biol. Cell. 2004; 15: 4382-4394Crossref PubMed Scopus (89) Google Scholar). Mutations that affect specific Chlamydomonas IFT particle proteins also result in flagellar assembly phenotypes. For example, deletion of the complex B genes, IFT88 (4Pazour G.J. Dickert B.L. Vucica Y. Seeley E.S. Rosenbaum J.L. Witman G.B. Cole D.G. J. Cell Biol. 2000; 151: 709-718Crossref PubMed Scopus (863) Google Scholar) and IFT52 (25Brazelton J.B. Amundsen C.D. Silflow C.D. Lefebvre P.A. Curr. Biol. 2001; 11: 1591-1594Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 26Deane J.A. Cole D.G. Seeley E.S. Diener D.R. Rosenbaum J.L. Curr. Biol. 2001; 11: 1586-1590Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), 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 (27Pedersen L.B. Miller M.S. Geimer S. Leitch J.M. Rosenbaum J.L. Cole D.G. Curr. Biol. 2005; 15: 262-266Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). As in Chlamydomonas, IFT mutants in other ciliated organisms are also characterized by defects in ciliary assembly (13Scholey J.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 423-443Crossref PubMed Scopus (337) Google Scholar). 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 (12Cole D.G. Traffic. 2003; 4: 435-442Crossref PubMed Scopus (195) Google Scholar). These domains include tetratricopeptide repeats (28Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (552) Google Scholar), WD-40 repeats (29Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar), coiled-coil domains (30Adamson J.G. Zhou N.E. Hodges R.S. Curr. Opin. Biotechnol. 1993; 4: 428-437Crossref PubMed Scopus (95) Google Scholar), and novel degenerate repeats termed WAA repeats found only in IFT particle proteins (27Pedersen L.B. Miller M.S. Geimer S. Leitch J.M. Rosenbaum J.L. Cole D.G. Curr. Biol. 2005; 15: 262-266Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 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 (31Qin H. Diener D.R. Geimer S. Cole D.G. Rosenbaum J.L. J. Cell Biol. 2004; 164: 255-266Crossref PubMed Scopus (266) Google Scholar). 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 (32Iomini C. Babaev-Khaimov V. Sassaroli M. Piperno G. J. Cell Biol. 2001; 153: 13-24Crossref PubMed Scopus (180) Google Scholar). 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 (33Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3471) Google Scholar). 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 (31Qin H. Diener D.R. Geimer S. Cole D.G. Rosenbaum J.L. J. Cell Biol. 2004; 164: 255-266Crossref PubMed Scopus (266) Google Scholar). 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. 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 (34Gorman D.S. Levine R.P. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1665-1669Crossref PubMed Scopus (1286) Google Scholar) 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 (17Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar), 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 (17Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar). 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) (35Asamizu E. Nakamura Y. Sato S. Fukuzawa H. Tabata S. DNA Res. 1999; 6: 369-373Crossref PubMed Scopus (156) Google Scholar, 36Asamizu E. Miura K. Kucho K. Inoue Y. Fukuzawa H. Ohyama K. Nakamura Y. Tabata S. DNA Res. 2000; 7: 305-307Crossref PubMed Scopus (103) Google Scholar). 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 (27Pedersen L.B. Miller M.S. Geimer S. Leitch J.M. Rosenbaum J.L. Cole D.G. Curr. Biol. 2005; 15: 262-266Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 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. 37Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.37-7.52Google Scholar. 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. (38Witman G.B. Carlson K. Berliner J. Rosenbaum J.L. J. Cell Biol. 1972; 54: 507-539Crossref PubMed Scopus (333) Google Scholar) 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 × g for 2 min at 25 °C. Cell pellets were resuspended into 4–6 liters of fresh M1 medium (39Sager R. Granick S. Ann. N. Y. Acad. Sci. 1953; 56: 831-838Crossref PubMed Scopus (329) Google Scholar) 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 × 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 × 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 × 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 × 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 × 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 × 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 × 10–7 cm2 s–1. Gel filtration standards used included thyroglobulin (2.6 × 10–7 cm2 s–1), apoferrition (3.24 × 10–7 cm2 s–1), alcohol dehydrogenase (4.76 × 10–7 cm2 s–1), and bovine serum albumin (6.3 × 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 (40Cantor C.R. Schimmel P.R. Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function. W. H. Freeman and Co., San Francisco, CA1980: 605-607Google Scholar),M=SRT/D(1−vρ)eq.1 where R is the gas constant, T is temperature in Kelvin, ν is the partial specific volume of the protein, ρ 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 (17Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar), 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 (41Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 70-76Google Scholar). 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 2× SDS sample buffer. Immunoprecipitates were separated on 4.0, 5.0, 6.0, or 7.5% SDS-polyacrylamide gels (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) 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 (43Clauser K.R. Baker P.R. Burlingame A.L. Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (981) Google Scholar). 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; Kazusa DNA Research Institute, Kisarazu, Chiba, Japan) (35Asamizu E. Nakamura Y. Sato S. Fukuzawa H. Tabata S. DNA Res. 1999; 6: 369-373Crossref PubMed Scopus (156) Google Scholar, 36Asamizu E. Miura K. Kucho K. Inoue Y. Fukuzawa H. Ohyama K. Nakamura Y. Tabata S. DNA Res. 2000; 7: 305-307Crossref PubMed Scopus (103) Google Scholar), 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 (31Qin H. Diener D.R. Geimer S. Cole D.G. Rosenbaum J.L. J. Cell Biol. 2004; 164: 255-266Crossref PubMed Scopus (266) Google Scholar). 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 (27Pedersen L.B. Miller M.S. Geimer S. Leitch J.M. Rosenbaum J.L. Cole D.G. Curr. Biol. 2005; 15: 262-266Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 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 (44Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2077) Google Scholar). 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 × 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 × 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 × 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 a" @default.
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• W2032141866 title "Characterization of the Intraflagellar Transport Complex B Core" @default.
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