The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a Repeat region with tandemly organized repeat sequences. In this study, we focused on the variations of the Repeat region, and searched for MAP4 isoforms with diverse Repeat region organizations. We successfully isolated four types of MAP4 cDNAs, which differed from each other in both the number and the arrangement of the repeat sequences, from a single source (bovine adrenal gland). To examine the functional differences among the isoforms, we prepared the microtubule-binding domain polypeptides of three of the four isoforms, and examined their activities. The isoform fragments showed similar degrees of microtubule assembly promoting activity and microtubule binding affinity. This result suggested that the Repeat region variation is not important for the control of microtubule dynamics, which is believed to be the main function of MAPs. On the other hand, the microtubule bundle-forming activity differed among the isoform fragments. The bundle formation was augmented by increasing the number of repeat sequences in the fragments. Based on these results, we propose the hypothesis that the role of the MAP4 isoforms is to regulate the surface charge of microtubules.

1990) J. Biol. Chem. 265, 13849±13855]. To compare the microtubule-binding mechanisms of the three MAPs, we performed a quantitative competition analysis using the three MAPs and the microtubule-binding domain fragment of MAP4 (PA 4 T fragment). The two-cycled microtubule protein fraction from bovine brain contains MAP1, MAP2, MAP4, and tau. When an excess of the PA 4 T fragment was added to the microtubule protein fraction, MAP4 and tau were completely released from the microtubules, while MAP1 remained bound. MAP2 was only partially released from the microtubules. The competition between MAP2 and MAP4 was further analyzed using purified MAP2, the PA 4 T fragment, and tubulin. About half of the MAP2 was still bound to the microtubules, even in the presence of an excess amount of the PA 4 T fragment. The microtubule-binding mechanisms of MAP2 and MAP4 seem to be different, in spite of their similar primary structures.Keywords: cytoskeleton; microtubule; microtubule-associated proteins; tubulin.Microtubules are found in all eukaryotic cells, and are essential in cell division, cell morphology, cell motility, and cytoplasmic organization. Microtubules consist of tubulin, the major component, and several types of microtubule-associated proteins (MAPs). MAPs are believed to play an important role in the regulation of microtubule formation and stabilization in vivo. To clarify the mechanisms of various microtubuledependent cellular functions, it is necessary to examine the MAP±microtubule interaction in detail.To date, several MAPs (MAP1, MAP2, tau, and MAP4) have been well characterized at the biochemical level [1±4]. Early reports showed that these MAPs can be divided into two domains, a projection domain and a microtubule-binding domain [5±7]. Recent studies of the overall primary structures of MAP1 [8,9] , MAP2 [10±12], tau [13±17], and MAP4[18±20] have revealed that the microtubule-binding domains of MAP2, tau, and MAP4 are structurally similar, while that of MAP1 is totally distinct. The microtubule-binding domains of MAP2, MAP4, and tau are divided into three subdomains: a proline rich (Pro-rich) region, an assembly promoting (AP) sequence region, and a short hydrophobic tail region [18]. Based on sequence similarity, it is believed that the three MAPs form a MAP subclass, and that their microtubule-binding mechanisms are similar.In support of this, MAP2 and tau [21,22] or MAP4 and tau [23] compete for their binding to microtubules. It was also reported that the microtubule-binding properties of MAP1 are different from those of the other MAPs [24±27]. On the other hand, detailed biochemical analyses, using truncated fragments of MAP2 [28,29], MAP4 [30,31], and tau [32,33], have revealed some differences in the binding mechanisms. For example, the stoichiometry of binding between the MAP2 microtubule-binding domain and the tubulin dimer was about 1 : 1 [29], while that between the MAP4 microtubule-binding domain and the tubulin dimer was about 2 : 1 [31].The abovementioned competition analyses [21±27] were too...

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