Goode_and_Feinstein_1994 - Identification of a Novel...

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Unformatted text preview: Identification of a Novel Microtubule Binding and Assembly Domain in the Developmentally Regulated Inter-repeat Region of Tau Bruce L. Goode and Stuart C. Feinstein Neuroscience Research Institute and Division of Molecular, Cellular, and Developmental Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 Abstract. Tau is a developmentally regulated microtubule-associated protein that influences microtu- bule behavior by directly associating with tubulin. The carboxyl terminus of tan contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previ- ously thought to function as "linkers," Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys 265 and lys TM, with a lesser contribution from lys TM. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed ar- ray of weak interactions thought to underlie the association between 18-amino acid repeats and micro- tubules (Butner, K. A., and M, W. Kirsclmer. J. Cell Biol. 115:717-730). Moreover, Competition assays sug- gest that the R1-P,2 IR associates with microtubules at tubuIin site(s) distinct from those bound by the re- peats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence- dependent manner. Since the RI-R2 IR is specifically expressed in adult tan, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tan molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons. T Au is a developmentally regulated family of micro- tubule-associated proteins (MAP) 1 specifically ex- pressed in neurons (Binder, 1985; for recent reviews see Lee, 1990; Wiehe et al., 1991). One-dimensional gel electrophoresis reveals two bands of apparent molecular mass 48-50 kD in fetal brain and four to six bands (48-67 kD) in adult brain (Cleveland et al., 1977a; Mareck et al., 1980; Francon et al., 1982; Ginzburg et al., 1982; Drubin et al., 1984a; Couchie and Nunez, 1985; Goedert et al., 1990). In addition, several high molecular mass forms oftau (119-125 kD) are expressed in the peripheral nervous system and in some neuronally derived cell lines (Drubin et al., 1985, 1988; Georgieff, 1991; Oblinger et al., 1991; Couchie 1985, 1988; Georgieff, 1991; Oblinger et al....
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Goode_and_Feinstein_1994 - Identification of a Novel...

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