Configuration of Flagellar Microtubule Subunits

0 Department of Biology, Biological Research Laboratories, Syracuse University , Syracuse, Neiv York 13210 , U.S.A. , and Department of Cell Biology, Centro de Investigation del I. P.N., Mexico 14, D.F F. D. WARNER AND I. MEZA SUMMARY Microtubule protofilaments and their subunits isolated from sperm flagellar doublet tubules of the sea urchin Strongylocentrotus purpuratus were examined by analytical biochemistry and high-resolution negative staining electron microscopy. All microtubule (tubulin) fractions show 2 polypeptide bands (a and /? tubulins) in an approximate 1:1 ratio on urea-polyacrylamide gel electrophoresis. Heat (37 C)-solubilized microtubules yield a protein fraction containing the tubulin dimer of molecular weight 115 000 Daltons. The dimeric tubulin subunit, as seen in the electron microscope, has an overall size of about 35 x 8 nm and appears to have the configuration of a figure 8 because of stain penetration into the centre of each of its 2 halves (figure os). Isolated protofilaments (35 03 nm thick) can each be resolved into 2 subfilaments (1-7 0-2 nm thick). The 2 subfilaments have periodic lateral associations resulting in the basic 4-nm subunit repeat (figure o) along the protofilament. Examination of collapsed and solubilizing protofilaments shows the figure 8 (dimeric) subunits separating at random along the protofilaments. We conclude that the tubulin dimer must be composed of either elongated or bilobed monomers which result in the figure 8 configuration and hence the 2-stranded appearance of the protofilaments. Feit, Slusarek & Shelanski, 1971; Meza, Huang & Bryan, 1972; Olmsted, Witman, CONFIGURATION Microtubules comprise a class of proteins termed tubulins that show similar biochemical and physicochemical properties. Microtubule protein was first extensively characterized from sea-urchin sperm flagella by Shelanski & Taylor (1967, 1968) who observed a single polypeptide band on urea-polyacrylamide gel electrophoresis. The protein, as a monomer, had a molecular weight of approximately 60000 Daltons. The microtubule protein dimer obtained from the central pair tubules showed both colchicine and specific nucleotide (GTP) binding activity. Subsequently, Renaud, Rowe & Gibbons (1968), studying doublet tubules from Tetrahymena cilia, observed that the microtubule protein separated as 2 close bands on urea-polyacrylamide gels. Stephens (1970) later isolated 2 proteins from the doublet tubules of Strongylocentrotus sperm flagella and suggested that only one occurred in each of the A and B subfibres of the outer doublet microtubules. Recently, however, several reports have appeared which clearly show that single microtubules, regardless of their source, contain in equal amounts both of the 2 tubulins termed either a,ft or 1,2. The tubulins differ in molecular weight by 30006000 Daltons and have similar amino acid compositions (Bryan & Wilson, 1971; attempts at structurally characterizing either the molecular or morphological subunits of microtubules have not been successful. The main morphological subunit of a microtubule is the 3-5-nm-thick protofilament, of which most single tubules have 13. The morphological subunit of a protofilament, as viewed by negative-staining electron microscopy (Grimstone & Klug, 1966) and by X-ray diffraction (Cohen, Harrison & Stephens, 1971), is a ~4-nm spherical particle, although larger orders of repeating structure are sometimes detectable along the filaments. In part by analogy with Gand F-actin, the 4-nm spherical subunit was thought to correspond to the tubulin monomer of average molecular weight 55000 Daltons. The tubulin dimer was thus thought to consist of 2 of the 4-nm subunits, which agrees reasonably well with the available physicochemical data on the tubulin molecule (Shelanski & Taylor, 1968). Utilizing outer doublet microtubules obtained from the sperm flagella of the sea urchin Strongylocentrotus purpuratus, we herein present data for a new and different morphological interpretation of the tubulin dimeric subunits and their association into Flagellar isolation and doublet microtubule preparation Flagella were obtained from the sea urchin Strongylocentrotus purpuratus following the methods described by Stephens (1970) and modified according to our needs. Sea urchins were shed by injection of 0-5 M KC1 into the body cavity. Sperm was collected in seawater, centrifuged down and resuspended in seawater containing io~* M EDTA. Sperm tails were detached from the heads using a Waring Blender and the 2 components separated by differential centrifugation. Isolated flagella were resuspended in a solution containing 1 % Triton X-100, 30 mM Tris-HCl, 3 mM MgCl, and O'i% mercaptoethanol at pH 8-o, gently homogenized with a glass pestle and centrifuged at ioooog. This results in a pellet of demembranated 9 + 2 flagellar axonemes. The axonemes were then extracted twice for 15 min each in a solution containing o-6 M KC1, 10 mM Tris-HCl and o-i % mercaptoethanol at pH 8-0 and centrifuged at ioooog. The extracted pellet was washed twice with 10 mM Tris-HCl at pH 8-o by resuspension and centrifugation. The final pellet of doublet microtubules was resuspended in either 10 mM Tris-HCl or 10 mM sodium phosphate buffer at pH 6-75. The KC1 extraction procedure preferentially solubilizes the central microtubules and many of the secondary structures of the axoneme, resulting in a clean preparation of outer doublet microtubules containing in excess of 90% tubulin, as illustrated in Fig. 1. Protein concentrations for all subsequent procedures were determined by the Lowry method (Lowry, Rosebrough, Farr & Randall, 1951). Solubilized protein is defined here as that not recoverable in a pellet by centrifugation at 100000 g for 60 min. Doublet microtubules were solubilized by the thermal fractionation procedure (Stephens, 1970). The doublets were resuspended in a solution containing 10 mM sodium phosphate at pH 6-75, heated to either 37 C or 50 C for 2-10 min and then centrifuged at 100000 g for 60 min. Heat treatment preferentially solubilizes the B subfibre or microtubule: 37 CC releases approximately 30 % of the tubulin into solution, while 50 C increases the yield to about 60 %. Reduction and carboxymethylation Following the method reported by Renaud et al. (1968), intact doublets or solubilized fractions were reduced in a solution containing 0 1 M mercaptoethanol, 8 M urea, o-i % E D T A and 0-35 M Tris-HCl at pH 8-8. For acetylation, 9 vol. of the reduced protein solution were mixed with 1 vol. of acetylating solution containing 1 1 M iodoacetic acid, 8 M urea and 1 M Tris base. T h e reduced and carboxy-methylated protein was then dialysed against 10 mM sodium phosphate - 10 rnM KC1 at pH 6 7 5 , at 4 C overnight and centrifuged at 100000 g for 60 min. T h e supernatant contains in excess of 90 % of the original doublet tubulin and samples were electrophoresed as indicated below. Urea-polyacrylamide gel electrop (...truncated)