Purified native microtubule associated protein MAP1A: Kinetics of microtubule assembly and MAP1A/tubulin stoichiometry

Abstract

In a recent study, we have shown that sulfonate buffers affect microtubule assembly and alter microtubule protein composition (Pedrotti et al., 1993). In particular, we noted that PIPES buffer leads to removal of MAP1 from the microtubule surface without affecting the association of MAP2 with microtubules. This observation has been exploited to develop a simple purification procedure for MAP1A using twice-cycled microtubule protein prepared from whole bovine brain. A single chromatographic step on an ion-exchange column results in > 90% pure MAP1A. Using purified MAP1A, we now show that MAP1A (a) binds in a dose-dependent manner to unpolymerized tubulin and assembled microtubules, (b) binds 13-15 mol of tubulin dimers in assembled microtubules, (c) promotes both nucleation and elongation of tubulin, and (d) promotes incorporation of tubulin dimers at low GTP concentrations and of tubulin dimers and oligomers at high GTP concentrations. MAP1A lowers the critical concentration for assembly, and MAP1A-promoted incorporation of dimers has an association rate constant (K+1) of 39.3 x 10(6) M-1s-1 and a dissociation rate constant (K-1) of 15 s-1; both constants are about 2-3-fold higher compared with MAP2.

Microtubule associated protein 1B (MAP1B) promotes efficient tubulin polymerisation in vitro

“…2A), the stoichiometry MAP1B and MAP2 to tubulin dimers was roughly 1 mol MAP1B: 13 mol tubulin dimers, and 1 mol MAP2:8 mol tubulin dimers. The MAP2 stoichiometry was similar to that previously reported [11]. Electron microscopy confirmed that 'normal' microtubules were present in samples containing MAP1B (Fig.…”

Modulation of microtubule shape in vitro by high molecular weight microtubule associated proteins MAP1A, MAP1B, and MAP2

“…The difference in length of MAP1B and MAP2 microtubules probably reflects the lower level of nucleation induced by MAP1B as the final level of polymerisation is similar to that observed with MAP2 (see also [24]). On the other hand, MAP1A microtubules are shorter than MAP2 microtubules, the level of nucleation is similar for both proteins, but MAP1A exhibits a higher critical concentration for assembly [18]. Several microscopic fields, containing between 400 and 450 microtubules, were also analysed to determine the number of bendy or straight microtubules formed in the presence of each different MAP (Fig.…”

“…On the other hand, MAP1B microtubules are generally long and bendy and the protein is highly concentrated in growth cones [11] where splayed, looped, and bendy microtubules have been observed [1,2]. This morphology coupled with the fact that MAP1B is (a) less efficient at stabilising microtubules against depolymerising drugs [16], (b) promotes tubulin polymerisation in vitro more efficiently when compared with MAP2 and MAPIA [18,24], and (c) has a lower binding affinity to microtubules than that of other MAPs [12][13][14], suggests that MAP1B may play a specialised role in permitting the formation of dynamic and bendy microtubules in regions characterised by events requiring rapid morphological reorganisation [1,2].…”

“…Tubulin, MAP2, MAP1A and MAP1B were purified as described by Pedrotti and Islam [18,22]. Protein concentration was determined using Bio-Rad (Rockville Centre, NY) protein reagent and bovine serum albumin as standard.…”

“…MAP1A, which exhibits a significant sequence homology with MAP1B, binds to different sites on the microtubule compared with MAP1B and MAP2 both in vivo [17] and in vitro [18]. A novel acidic binding domain has been identified in MAP1A [19] and transfection studies, with a truncated form of MAP1A containing this site, in cell lines have shown that expression of MAP1A leads to microtubules becoming somewhat shorter and much more straight [19].…”