Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin.

A Banerjee,M C Roach,P Trcka,R F Ludueña

doi:10.1016/s0021-9258(19)40087-2

Abstract

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.

Highlights

We find that removal of the,&u isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPS), tau or MAP 2
Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules
Chromatography of Phosphocellulose-purified Tubulin on the Antibody-Sepharose Affinity Matrix-An affinity column was prepared by coupling the antibody SDL.3DlO with CNBractivated Sepharose

Summary

PROCEDURES

Lys-OH was synthesized by the BioSearch Corporation, San Rafael, CA. Except for the amino-terminal cysteine, the peptide has a sequence identical to that of the last amino acids in human &tubulin (Sullivan and Cleveland, 1986). Based on previous results (Banerjee et al, 1988), the unbound fraction should contain Both fractions were reduced and carboxymethylated and subjected to preparative electrophoresis (Laemmli, 1970) on gels containing 5.5%. Cell-free supernatants of six cell lines were tested by immunoprecipitation using protein A-agarose These six tested negative against peptides corresponding to the C termini of the @rvand flv subunits and tested positive to the peptide corresponding to the C terminus of flrrr as well as to a mixture of&rand @Iv-tubulins. The cell line SDL.SDlO bound best to protein A and was selected for growing in roller bottles and for subsequent purification by chromatography on protein A-agarose as previously described (Baneriee et al, 1988). Protein determination was done according to Lowry et al (1951)

RESULTS

PC u B PC U B PC U B

One possibility is that the antibody has a weak affinity for

DISCUSSION