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. 2000 Jan;11(1):201-15.
doi: 10.1091/mbc.11.1.201.

The Rib43a Protein Is Associated With Forming the Specialized Protofilament Ribbons of Flagellar Microtubules in Chlamydomonas

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The Rib43a Protein Is Associated With Forming the Specialized Protofilament Ribbons of Flagellar Microtubules in Chlamydomonas

J M Norrander et al. Mol Biol Cell. .
Free PMC article

Abstract

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins as tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1. 7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an approximately 4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.

Figures

Figure 1
Figure 1
SDS-PAGE of ribbons and Western blot analysis with anti-rib43a antibodies. (A) Sarkosyl-insoluble ribbons (20 μg/lane) from sea urchin Strongylocentrotus purpuratus sperm flagella and C. reinhardtii flagella, showing tubulin and the main components of S. purpuratus (tektin-B ≅ 51-kDa, tektin-C ≅ 47-, 77-, and 83-kDa polypeptides; tektin-A ≅ 53-kDa comigrates with tubulin) and of C. reinhardtii. Calibration masses are given at 89, 117, and 197 kDa. The C. reinhardtii protein rib43a was isolated, transferred to nitrocellulose, and used to raise polyclonal rabbit antibodies for subsequent cloning. (B) SDS-PAGE blots of Chlamydomonas axonemes (40 μg/lane) demonstrating the specificity of anti-rib43a antibodies. Lane 1, protein stain; lane 2, preimmune sera; lane 3, whole anti-rib43a antisera; lane 4, affinity-purified anti-rib43a antibodies; lane 5, affinity-purified anti-rib43aΔN64 antibodies (against the expression protein; see text). Calibration masses are given in kilodaltons; arrowheads indicate the position of the rib43a protein, with a nominal molecular weight of 46,000.
Figure 2
Figure 2
Northern blots of poly(A)+ RNA (5 μg/lane) isolated from Chlamydomonas before and after (30 min) deflagellation. (A) pBrib43a hybridizes with a 1.7-kb band, which is up-regulated after deflagellation. (B) pCRY1-1, a clone coding for the S14 ribosomal protein, which is not involved in flagellar assembly (Nelson et al., 1994), hybridizes with 0.9-kb bands of equal intensity before and after deflagellation. These results demonstrate that the up-regulation seen with pBrib43a is not due to unequal loading of RNA.
Figure 3
Figure 3
Western blot analysis of antibodies raised against the native Chlamydomonas ribbon protein rib43a (anti-rib43a) and against the bacterially expressed, truncated fusion protein from pBrib43aΔN64 (anti-rib43aΔN64). (A) SDS-PAGE blots of lysates from bacteria transformed with pBrib43aΔN64, as follows: lanes 1, 3, and 5, uninduced; lanes 2, 4, and 6, induced with IPTG. Lanes 1 and 2 were stained with Ponceau S; lanes 3 and 4 were stained with anti-rib43aΔN64; and lanes 5 and 6 were stained with anti-rib43a. The IPTG-induced fusion protein rib43aΔN64 (arrowheads) is recognized both by the antibody made against it (anti-rib43aΔN64, lane 4) and by anti-rib43a (lanes 6). (B) SDS-PAGE blots of whole Chlamydomonas cells (Cells, ∼80 μg) and the sequential fractionation of flagella (Fla, 60 μg) into axonemes (Axo, 40 μg) and ribbons (Rib, 20 μg). Lanes 1, 3, 5, and 7, stained with Ponceau S; lanes 2, 4, 6, and 8, stained with affinity-purified anti-rib43aΔN64 antibodies (against the bacterially expressed fusion protein). Anti-rib43aΔN64 antibodies continue to stain the rib43a protein (arrowheads) that is retained in the purified ribbons after fractionation of flagella and axonemes. Only faint staining of rib43a could be seen in the original, freshly stained blot of whole cells; the apparent bands seen here are attributable to faint, uneven background across the nitrocellulose sheet.
Figure 4
Figure 4
Predicted sequence of rib43a and comparison with other sequences in the database. The largest open reading frame of the pBrib43a cDNA consists of 1612 bp encoding rib43a, the 367-amino-acid protein from Chlamydomonas ribbons with a molecular weight of 42,638. The predicted sequence of rib43a is compared with sequences (ESTs) from T. cruzi and tumors of H. sapiens. Compared with rib43a, identical and conservatively substituted residues are shown in black and gray, respectively. Between rib43a and the Trypanosoma EST there is 35% identity and 46% similarity (similarity = identities + conservative substitutions) with no gaps in the sequences; between rib43a and the human tumor ESTs these values are 28 and 37% for EST1 and 26 and 38% for EST2 (with only one gap). Several blocks of amino acid residues are identically conserved between rib43a and the trypanosome EST (e.g., RVGDDD) and between rib43a and both of the human tumor ESTs (e.g., KGMT). Note that rib43a has no cysteine residues. The references of the clones are as follows: rib43a (this report; GenBank accession numbers AF196576 for the cDNA clone and AF196577 for the genomic clone); Trypanosome (GenBank accession number AI080816); human EST1 (GenBank accession number AI890123) from moderately differentiated, endometrial adenocarcinoma, three pooled tumors; and human EST2 (GenBank accession number AI671905) from pooled germ cell tumors.
Figure 5
Figure 5
Predicted coiled-coil structure of Chlamydomonas rib43a, using the CoilScan program (Lupas et al., 1991; Lupas, 1996). Probability of the formation of coiled coil (ordinate) is plotted along the polypeptide chain from N to C terminus (abscissa).
Figure 6
Figure 6
The structure of the 4.4-kb wild-type genomic fragment containing the RIB43a gene. Boxed areas indicate sequence contained in the cDNA clone pBrib43a; shaded boxes designate the largest open reading frame. Comparison of the cDNA length (1612 bp) with the estimated size of the RIB43a transcript (∼1700 bases by Northern blotting; Figure 2) puts the transcription initiation site at ∼1051 bases; the predicted translation start codon is located at base 1244. There are 11 potential tub boxes (at least 70% identical to the consensus sequence GTTCSAAGGC; Davies and Grossman, 1994) located at bases 48, 136, 318, 397, 425, 535, 542, 694, 783, 879, and 953; these sequence elements are thought to be important for regulated expression of flagellar transcripts. Potential TATA boxes are located at 1007 bases (TTTATGA), 1009 bases (TATGATA), 1012 bases (GATAATT), and 1046 bases (TACACAT). The translation stop codon is present at base 3808. A polyadenylation site (TGTAA) appears at base 4202; the start of the poly(A) tail on our cDNA corresponds to base 4220 of the genomic sequence.
Figure 7
Figure 7
Southern blot analysis of gene copy number. The hydridization pattern of blots of genomic DNA digested with XhoI, SacI, and HindIII probed with pBrib43a agrees with the restriction patterns predicted from the pRIB43a sequence. This suggests there is a single RIB43a gene. Size standards are indicated on the right.
Figure 8
Figure 8
Negative stain and immuno-EM of Chlamydomonas ribbons. (A and B) Preparation of purified ribbons, negatively stained with 1% uranyl acetate. Ribbons are homogeneously composed of three protofilaments (three lines in B). Some ribbons are straight and rigid, and others are twisted and curled; some ribbons form pairs or sheets. A rare, contaminating A-microtubule is included for size comparison and to show the ribbon emerging from its end (arrow). (C–E) Ribbons stained with affinity-purified, rabbit anti-rib43aΔN64 antibodies, followed by 5-nm colloidal gold-conjugated goat anti-rabbit IgG. Ribbons are sparsely and randomly labeled along their length and at their ends (arrows). Many apparent background gold particles are actually due to label on short pieces of ribbons and on short, thin fibrils (arrowheads). (F) Control showing absence of staining when primary antibody is omitted. Bars, A, C, D, and F, 0.2 μm; B and E, 100 nm.
Figure 9
Figure 9
Immunofluorescence microscopy of whole cells and basal body–flagellar complexes. (A–D) Cells stained with affinity-purified rabbit anti-rib43aΔN64 antibody, followed by Texas Red-conjugated goat anti-rabbit IgG. (E) Phase-contrast image; (F) lack of staining with preimmune serum. (G and H) Immunofluorescence of isolated basal body–flagellar apparatuses stained with anti-rib43aΔN64. These results demonstrate that anti-rib43a antibodies specifically stain basal bodies and flagella in a punctate manner from base to tip. In addition, the anti-rib43a antibodies stained structures corresponding to the proximal portion of the four rootlet microtubules (G and H). Bars, A–F, 5 μm; G and H, 5 μm.

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