Revised Manuscript Received May 16, 1994• abstract: The role of the neuronal microtuble-associatedprotein r has been studied by generating a series of r constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N-or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of themutant r proteins with microtubules was judged byseveral independent methods,(i) Direct binding assays between r and taxol-stabilized microtubules yield dissociation constants and stoichiometries,(ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of t to promote microtubule nucleation, elongation, and bundling in bulk solution,(iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as “jaws” for r on the microtubule surface (the proline-rich domain P upstream of the repeats andthe “fifth” repeat R/downstream). Such combinations make r’s binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, ie, when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R'causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the r isoforms are not “bundling proteins” in the proper sense. In spite of the widerange of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 r/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.(a) MAPs and Microtubule Assembly. Microtubule-as-sociated proteins (MAPs) 1 derive their name from the fact that they copolymerize with microtubules through cycles of assembly and disassembly. This means that they bind to microtubules. More importantly, however, they also stimulate microtubule assembly and stabilize them, once formed. The tissue-and development-specificstabilization of microtubules is presumably one of the important functions of MAPs [for recent reviews, see Wiche et al.(1991), Chapin and Bulinski (1992), and Lee (1993)]. A number of MAPs have now been cloned and sequenced. Depending on sequence homology, they can be subdivided into several families. The best known family is that of the rM AP2-M AP4 proteins [for r, see Drubin et al.(1984), Leeet al.(1988), and Himmler et al.(1989); for MAP2, Lewis et al.(1988) and Kindler et al.(1990); for MAP4, Aizawa et al.(1990), West et al.(1991), and Chapin and Bulinski (1991)]. They derive from different genes but share several homologous «= 31-residue repeats near their C-termini that are involved in microtubule binding. MAP2 and r occur in the brain; MAP4 is ubiquitous. t Thisproject was supported by the Bundesministerium fur Forschung und Technologie and the Deutsche Forschungsgemeinschaft.* Corresponding author. Tel:(+ 49)(40) 8998-2810. Fax:(+ 49)(40) 891314.