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. 2003 Jan 2;22(1):70-7.
doi: 10.1093/emboj/cdg001.

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol

Santwana Kar  1 , Juan Fan, Michael J Smith, Michel Goedert, Linda A Amos
Affiliations

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol

Santwana Kar et al. EMBO J. .

Abstract

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.

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Figures

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Fig. 1. Comparison of differences in tau proteins. (A) Diagram of four-repeat tau (4R-tau) molecule. The N-terminal segment forms a projection from a microtubule when the rest of the molecule is bound (Hirokawa et al., 1988). R1–R4 repeat motifs are flanked by proline-rich (P1–P2) and C-terminal segments. 3R-tau is similar apart from lacking one of the repeat motifs. (B) Amino acid sequence of the four repeats in one-letter code. Mutation sites C291I, C322I and S305C are indicated. Boxed sequence (V275–S305) is absent from three-repeat tau (3R-tau). The sequence from α-tubulin (T361–L368) is an extra loop filling the equivalent of the taxol site. (C) Spectroscopic assay of microtubule assembly with 4R-tau and 3R-tau, each purified by two methods (see text).
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Fig. 2. Pelleting assays. (A and B) SDS–polyacrylamide gels showing proteins in pellets (P) and supernatants (S) when 10 µM tubulin dimer was incubated with increasing concentrations of either triple mutant 4R-tau (A) or wild-type 3R-tau (B). Tau runs anomalously above tubulin (Tu) on gels. Wild-type 4R-tau (see Figure 3) and the mutant (shown here and in Figure 5C) gave indistinguishable results. (C and D) Plots of pelleted tau measured by densitometry of gels such as those in (A) and (B). The points diverge first gradually, then much more rapidly, from the diagonal lines as increasing proportions of tau appear in the supernatant.
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Fig. 3. (A) Western blots of SDS gels, with lanes containing undigested tubulin (Tu) and subtilisin-digested tubulin (ST), incubated with four different anti-tubulin monoclonal antibodies specific to either α- or β-tubulin; the epitopes lie either at the very C-termini or closer to the N-termini. (B) SDS gels showing 3.0 µM wild-type 3R- and 4R-tau and 1.0 µM native MAP2 (M2) (Sigma) in pellets (P) and supernatants (S) when incubated with 10 µM S-tubulin (ST). To ensure its stability during digestion, ∼30 µM taxol was present with the S-tubulin. 1.0 µM MAP2 was also incubated with 10 µM control tubulin (Tu). (C) Pelleting assay to compare the binding to S-tubulin of 3R-tau purified by method 1 or method 2. (D) Binding of 3R-tau to normal tubulin in the absence or presence of kinesin motor domains (K). Results similar to those shown in (C) and (D) were obtained with 4R-tau.
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Fig. 4. Effect of taxol on tau binding. (A and B) SDS–polyacrylamide gels showing proteins in pellets (P) and supernatants (S) when 10 µM tubulin dimer was incubated with increasing concentrations of wild-type 3R-tau and 4R-tau in (A) the absence or (B) the presence of 20 µM taxol. Similar results were obtained for triple mutant 4R-tau, before and after labelling with nanogold (Figure 5). (C and D) Plots of pelleted WT tau measured by gel densitometry. A binding curve in the presence of 200 µM taxol is also included. (E) Scatchard plots of the results for 3R-tau showing the large change in the dissociation constant (inverse of the slopes of the lines) when excess tau is bound. In the absence of taxol, below 3 µM total tau there is so little in the supernatants that the slope of the strongly binding phase is not defined accurately by our data. There is even greater uncertainty for 4R-tau, which apparently binds more tightly (Supplementary data).
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Fig. 5. (A) Separation of free nanogold from labelled protein by gel filtration (red curve). (B) Fractions of the first peak were dotted onto nitrocellulose paper and stained to detect the presence of gold and protein. The second peak (free gold without protein) did not bind to the paper. (C) Binding assays of labelled and unlabelled triple mutant tau (4R-tauTM). The curve shown is for assembly with GMPCPP; results for GTP were similar.
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Fig. 6. (A) Electron micrograph of a frozen hydrated microtubule containing nanogold-labelled 4R-tauTM. Arrows indicate some of the gold particles that can be seen directly. (B) Model of a 15 kDa gold particle attached to a 50 kDa tubulin monomer (top left). In the image of a microtubule containing one labelled tau molecule per three to four tubulin dimers, a labelled monomer will usually be superimposed on an unlabelled monomer on the other side of the tube (lower left and centre). Noise in the image (right) makes some gold particles undetectable by eye. (C and D) Calculated diffraction patterns from electron microscope images of microtubules assembled with nanogold-labelled 4R-tauTM (C) without further decoration and (D) fully decorated with kinesin motor domains. The 4 nm layerline arises from the longitudinal spacing of tubulin monomers, and the 8 nm layerline from the binding of a kinesin molecule to each tubulin heterodimer (see Figure 7G).
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Fig. 7. Reconstructed images. (A–D) End-on views of microtubules without kinesin. (A) Pure tubulin (with GMPCPP). (B) Tubulin co-assembled with unlabelled tau. Tau difference density (at a lower cut-off level) is superimposed in blue on half of the map; some density appears on the inner (i) and outer (o) surfaces. (C) Tubulin co-assembled with labelled tau. The peak of the difference density due only to nanogold is superimposed in yellow. (D) Complete nanogold difference map [(C) minus (B)]. (E and F) Inside views of three- dimensional maps. (E) Three-dimensional version of (C). (F) As--sembled with labelled tau and decorated outside with kinesin (red). Peaks of the density difference between maps of kinesin-decorated microtubules containing labelled tau and without tau, are shown in yellow. (G) Outside view of kinesin-decorated microtubule assembled with gold-labelled tau. The difference map shows no density here at the level depicted in (F).
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Fig. 8. Background: pure tubulin map (three-dimensional version of Figure 7A). Superimposed: ribbon diagram of αβ-tubulin atomic structure (PDB 1JFF) (Löwe et al., 2001). Lα, α-tubulin loop (T361–L368) alone; T, taxol; N, bound nucleotide. Blue structure, model of bound tau repeats including PGGG loops; the asterisk indicates an inter-repeat region.
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References

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