Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8567-8571, September 1992 Cell Biology Primary structure of tektin Al: Comparison with intermediate- filament proteins and a model for its association with tubulin (basal body/centriole/dlia/flageila/microtubule) J. M. NORRANDER*, L. A. AMOSt, AND R. W. LINCK* *Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455; and tLaboratory of Molecular Biology, Medical Research Center, Hills Road, Cambridge, CB2 2QH, United Kingdom Communicated by M. F. Perutz, May 19, 1992 (receivedfor review January 22, 1992) ABSTRACT Tektins are proteins that form rilamentous MATERIALS AND METHODS polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to S. purpuratus gametes were collected, handled, and cultured those of intermediate-filament proteins. We report here the at 16'C, as described (14). At desired times, aliquots of sequence of a cDNA for tektin Al, one of the main tektins from eggs/embryos were isolated and frozen in liquid N2. Total Strongylocentrotus purpuratus sea urchin embryos. By hybrid- RNA and poly(A)+ mRNA were isolated (15). A Agtll cDNA ization analysis, tektin A mRNA appears maximally at cilio- expression library, constructed from blastulae (gift of T. L. genesis. The predicted structure of tektin Al (Mr 52,955) is a Thomas, Texas A & M, College Station, TX), was screened series of a-helical rod segments separated by nonhelical link- with polyclonal antibodies (16) against S. purpuratus sperm ers. The two halves of the rod appear homologous and are flagellar tektin A (11). The largest clone isolated, tekA10-2, probably related by gene duplication. Comparison of tektin Al contains a 2055-base-pair insert. Rescreening of the library with intermediate-filament proteins, including nuclear lamins, failed to uncover any larger tektin A clones. A second library reveals a low amino acid homology but similar molecular motif, was constructed from blastula mRNA in AZapI1 (Stratagene) i.e., pattern of helical and nonhelical domains. This study and screened with radiolabeled tekA10-2 (15). Several clones indicates that tektins are unique proteins but may be evolu- were isolated, including tekA5-8. Inserts were transferred to tionarily related to intermediate-filament proteins, and sug- the plasmids pUC118/119 (17), sequential deletion clones gests a structural basis for the interaction oftektins and tubulin were constructed (18), and dideoxy sequencing was per- in microtubules. formed (19). Sperm flagellar tektins were purified by N-lau- roylsarcosine/urea extraction and reversed-phase HPLC (3). Tektins A (-55 kDa), B (-51 kDa), and C (-47 kDa) were Tektin A, whose purity was checked by SDS/PAGE- originally identified as proteins comprising 2- to 3-nm- immunoblotting, was treated with N-chlorosuccinimide diameter filaments that remained insoluble after extraction of (NCS) (Aldrich), which cleaves primarily on the carboxyl sea urchin sperm flagellar microtubules (MTs) with chaotro- side of tryptophan (20) but may also act on histidine and pic solvents (1-3). Immunofluorescence microscopy demon- tyrosine. Protein was dissolved in 50-75% acetic acid and strated the presence of tektins in doublet MTs, and possibly treated with NCS for 1 hr at 370C (8 pmol of NCS per ml of in central pair MTs (4), and tektin-like components in cen- tektin per A280 unit). The reaction was terminated with excess trioles, centrosomes, and mitotic spindles (4-6). Immuno N-acetylmethionine. After evaporation to dryness, samples electron microscopy suggested that tektins formed extended were dissolved in SDS sample buffer. Peptides were sepa- polymers in the walls ofciliary and flagellar MTs (2). Tektins rated by SDS/PAGE and transferred to Immobilon-P (Mil- probably interact directly with tubulin in the MT wall, since lipore) (21), which was stained with Serva Blue R and dried. N-lauroylsarcosine detergent extraction of cilia and flagella Excised bands were sequenced by automated Edman degra- yielded stable ribbons of three protofilaments composed of dation on an Applied Biosystems 470 peptide sequencer by tubulin and tektins in a molar ratio of -2:1 (1-3, 7). We have E. D. Eccleston, Microchemical Facility, University of Min- determined the primary structure of tektin A, in order to nesota. Standard data bases were searched with the program better understand its structure and function in MTsA FASTA (22). For detailed sequence comparison, the DIAGON Tektins appear to be similar to intermediate-filament pro- program (23) was used; homology scores (using the Dayhoff teins (IFPs) in terms of their relative insolubility, molecular score matrix, based on observed substitution frequencies for weights, and fibrous, a-helical structure (1). Peptide map- residues in related proteins) were calculated to compare pairs ping, amino acid analysis, and immunological studies showed of residues, plus flanking sequences spanning 49 total resi- that the three main tektins were related to each other but not dues. The program calculates a "double matching probabil- to tubulin (2, 3) and substantiated the similarity between ity", i.e., that of obtaining a particular score, given two tektins and IFPs (3, 8, 9). We have begun to examine this infinitely long sequences of the same composition as those relationship more closely by characterizing a cDNA for tektin being compared. Scores with a probability of occurring by A from sea urchin embryos, obtained by screening a Agtll chance alone of <10-4 were plotted. Secondary structure library from Strongylocentrotus purpuratus blastulae with was predicted (24) with the program ANALYSEP (25). antibodies against flagellar tektin A. Immunofluorescence microscopy (10) and immunoblotting analysis (11) have shown that blastula cilia contain tektins that appear identical RESULTS to sperm flagellar tektins. Furthermore, a ciliary MT protein Two cDNA clones were fully sequenced. By several criteria of -55 kDa is synthesized de novo at the onset ofciliogenesis (below), clone tekA5-8 includes the complete coding se- in blastulae (12), and this has been identified as tektin A (13). Abbreviations: IFP, intermediate-filament protein; MT, microtu- The publication costs of this article were defrayed in part by page charge bule; NCS, N-chlorosuccinimide. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M97188). 8567 Downloaded by guest on September 27, 2021 8568 Cell Biology: Norrander et al. Proc. Natl. Acad. Sci. USA 89 (1992) quence for tektin, A, and tekA10-2 includes 70%o of the 1 2 3 4 5 6 7 8 9 10 11 12 coding sequence. Within their coding regions they show ... .... .... >97% nucleotide and >99%o amino acid identity; however, differences in the noncoding regions suggest that tektin A belongs to a multigene family. The results for tekA5-8, which 5.0, we have termed tektin Al, are shown in Fig. 1. The predicted 4.00- molecular weight of 52,955, acidic pI, amino acid composi- tion, and a-helical content all compare favorably with pre- - 2.75 vious experimental measurements for the tektin A protein 34 - M from sea urchins (1, 3, 11). ax 2.0 Direct evidence that tekA5-8 encodes tektin A was ob- (0 f.. .0 *= tained by partial amino acid sequencing of the flagellar form 0 of tektin A. Since the amino termini of SDS/PAGE-purified i2 1.oD. tektins are blocked, fragments of tektin A were obtained by cleavage with NCS. By SDS/PAGE, discrete peptide bands corresponded in molecular weight to predicted fragments that could be generated by complete and incomplete cleavage of tektin A at tryptophans (data not shown), corroborating the positions of the four tryptophans. Moreover, 12.5- and 26- kDa fragments yielded the following N-terminal sequences respectively (-, undetermined residues; alternative residues in parentheses): FIG. 2. Appearance of tektin mRNA during S. purpuratus em- bryogenesis. Eggs were fertilized and cultured. At selected times, AEFS(V)N(H)DNVVRAE-E-L(D)ADQQD(L)--LID-I(A)LTD, poly(A)+ mRNA was isolated, resolved, and probed with 32P-labeled cDNA clone tekA10-2. Lanes: 1, unfertilized eggs; 2, 4-cell stage; 3, A(ED)EFS(V)-DNVVRAE---LA-Q(V)Q(D) 32-cell stage; 4, 7.5 hr; 5, 9 hr; 6, 10.5 hr, beginning of ciliogenesis; 7, 12 hr, 50%o rotating blastulae; 8, 13.75 hr, hatched and swimming; The two sequences, probably identical because the 26-kDa 9, 17.5 hr; 10, 25.5 hr. late blastula; 11, 44.5 hr, early gastrula; 12, 52.5 peptide is partially cleaved at His-374 or -377, confirm 20-30 hr, gastrula. A major band of2750 bases is labeled maximally in lane residues, starting from Ala-273. A secondary sequence ob- 7; weaker bands are present earlier. Tektin A mRNA may have been tained from the 12.5-kDa band (a unique, comigrating frag- degraded in samples obtained after ciliogenesis. ment), KF-LN(H)REIEDMI(D), matches :12 residues of a predicted fragment generated by NCS cleavage at Trp-128 Taken together, the predicted features of clone tekA5-8, its and Trp-230. Clones tekA5-8 and tekA1-2 hybridize with a mRNA expression during ciliogenesis, and its coincidence 2750-base embryonic mRNA, which reaches peak levels with partial amino acid sequence in the native protein estab- during ciliogenesis (Fig. 2), when tektin A is maximally lish that the clone codes for tektin A. synthesized (12, 13). These observations indicate that both The deduced amino acid sequence oftektin A was analyzed clones encode tektin A, rather than a cytoplasmic IFP or a for secondary structure and internal repeats. Seventy percent nuclear lamin. We also noted a low level of mRNA hybrid- of the central segment (residues 87-432, referred to here as izing to tektin clones at earlier points after fertilization (lanes the rod) is strongly predicted to be a-helical (Fig.