The Interaction of TOGp with Microtubules and Tubulin

Abstract

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned fulllength TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The Cterminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.Microtubule assembly is regulated in cells to generate a relatively stable interphase microtubule array or the much more dynamic microtubules of the mitotic spindle. In either cell cycle stage, the major pathway of microtubule turnover is dynamic instability, where microtubules exist in persistent phases of growth or shortening with the abrupt transitions, termed catastrophe and rescue, between these phases (1). Several classes of microtubule assembly regulators have been identified that can be broadly classified as microtubule stabilizers (e.g. tau, MAP2, 1 or MAP4) or destabilizers (e.g. XKCM1 or oncoprotein 18; reviewed in Refs. 2 and 3). Together, the activities of these accessory proteins generate the dynamic microtubules observed in vivo (reviewed in Refs. 3 and 4).The stabilizing MAP, XMAP215, was initially isolated based on its preferential promotion of microtubule plus end assembly rates (5). Remarkably, this protein speeds the microtubule plus end growth rate by 7-10-fold, primarily through an increase in the apparent on-rate constant (5, 6). In contrast, other stabilizing MAPs, such as tau or MAP2, modestly increase growth rates ϳ2-fold at both microtubule ends, primarily through a decrease in the off-rate constant (7-9). More recent studies have demonstrated that the plus end stabilizing activity of XMAP215 can also counterbalance the catastrophe-promoting activity of XKCM1 (10). The mechanisms responsible for assembly promotion and catastrophe protection by XMAP215 are not known.Given the potent and unique effects of XMAP215 on microtubule assembly in vitro and in Xenopus egg extracts (5, 6, 10, 11), it is not surprising that a number of homologs have been identified in...

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The Microtubule-associated Protein Tumor Overexpressed Gene Binds to the RNA Trafficking Protein Heterogeneous Nuclear Ribonucleoprotein A2

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