Systematic Identification of Tubulin-interacting Fragments of the Microtubule-associated Protein Tau Leads to a Highly Efficient Promoter of Microtubule Assembly

Caroline Fauquant,Virginie Redeker,Isabelle Landrieu,Jean-Michel Wieruszeski,Dries Verdegem,Olivier Laprévote,Guy Lippens,Benoît Gigant,Marcel Knossow

doi:10.1074/jbc.m111.223545

Abstract

Tau is a microtubule-associated protein that stabilizes microtubules and stimulates their assembly. Current descriptions of the tubulin-interacting regions of Tau involve microtubules as the target and result mainly from deletions of Tau domains based on sequence analysis and from NMR spectroscopy experiments. Here, instead of microtubules, we use the complex of two tubulin heterodimers with the stathmin-like domain of the RB3 protein (T(2)R) to identify interacting Tau fragments generated by limited proteolysis. We show that fragments in the proline-rich region and in the microtubule-binding repeats domain each interact on their own not only with T(2)R but also with microtubules, albeit with moderate affinity. NMR analysis of the interaction with T(2)R of constructs in these two regions leads to a fragment, composed of adjacent parts of the microtubule-binding repeat domain and of the proline-rich region, that binds tightly to stabilized microtubules. This demonstrates the synergy of the two Tau regions we identified in the Tau-microtubule interaction. Moreover, we show that this fragment, which binds to two tubulin heterodimers, stimulates efficiently microtubule assembly.

Highlights

Involved in important functions such as long range organelle transport or mitotic spindle formation
Tau Fragments Belonging to the Pro-rich and MTBR Domains Interact with Tubulin in T2R—To identify Tau fragments that bind to tubulin, we have combined limited proteolysis of Tau and size exclusion chromatography (SEC) to select fragments that co-elute with the target
The objective of this work was the identification of Tau fragments that bind to tubulin or MTs to facilitate structural studies and to dissect the mechanism by which Tau promotes MT assembly

Summary

Introduction

Involved in important functions such as long range organelle transport or mitotic spindle formation. The second model relates to the importance of regions in the assembly domain for the effect of the different Tau isoforms on MT dynamics. It proposes that the initial binding of Tau to MTs is mediated by an MT-binding core within MTBR, whereas the flanking regions exert an isoform-specific regulation [28]. Regions in the proline-rich domain [23, 24] modulate MT binding and assembly, but they must be associated with repeats in the same fragment to have an effect. The contribution of basic residues has been investigated This identified several of them in the proline-rich domain and in ir as important for Tau interaction with MTs [24, 29]. Consistent with the basic nature of these residues, cross-linking suggests that the binding targets of repeat 1 and/or ir are the C-terminal acid tails of both ␣- and ␤-tubulin [30]

Objectives

Results

Conclusion