Microtubule-associated protein MAP1 promotes microtubule assembly in vitro

Abstract

Several proteins co-purify with tubulin through polymerization-depolymerization cycles. These proteins are called microtubule-associated proteins (MAPS), and some of them have been found to be really associated with cellular microtubules (review [l]). It is MAPS that are thought to be responsible for microtubule functions and interactions with other cellular components. Tubulin was prepared from bovine brain by an assembly-disassembly procedure [IO], modified as in [2], and separated from MAPS by chromatography on phosphocellulose (Whatman P-II) [ 1 l] in a buffer containing 50 mM imidazole-HCl (pH2,, 6.7), 50 mM KCl, 0.5 mM MgC12, 0.1 mM EDTA and 1 mM 2-mercaptoethanol (buffer A). Methods for MAP1 and unheated MAP2 purification were as in [9]. Three MAPS have already been purified. These are MAP2 [2-41 and 7 [5] from mammalian brain and the 210 OOOM, protein from HeLa cells [6,7]. These proteins were found to promote microtubule assembly in vitro. Their assembly-promoting activity is heatresistant. Little has been known about the activities of the heaviest of MAPS, MAP1 , which comprises a significant part of brain MAPS. There was only the report [8], which concluded that MAP1 does not possess any assembly-promoting activity. This conclusion was based on the fact that the removal of MAP1 from microtubule proteins had no effect on their polymerization. Tubulin was polymerized at 37°C in buffer A supplemented with 1 mM GTP and 1 mM EGTA. Polymerization was followed by monitoring the light scattering of the solution at 330 nm [ 121. The morphology of tubulin polymers was studied by electron microscopy after thin-sectioning or negative-staining of the samples with 1% aqueous uranyl acetate. For determination of the protein composition of the polymerization products they were pelleted by centrifugation at 200 000 X g through a cushion of buffer A containing 4 M glycerol and 1 mM EGTA, and the pellets were analysed by SDS gel electrophoresis.