Identification of a 34-kD Polypeptide as a Light Chain of Microtubule-associated Protein-1 (MAP-l) and Its Association with a MAP-1 Pepti le that Binds to Microtubules

We examined the association of a 34-kD light chain component to the heavy chains of MAP-1 using a monoclonal antibody that specifically binds the 34-kD component and labels neuronal microtubules in a specific and saturable manner. Immunoprecipitation of MAP1 heavy chains together with the 34-kD component by the antibody indicates that the 34-kD polypeptide forms a complex with MAP-1 heavy chains. Both major isoforms of MAP-1 heavy chains (MAP-1A and MAP-1 B) were found in the immunoprecipitate. Digestion of MAP1 with alphachymotrypsin and analysis of the chymotryptic peptides reveals a 120-kD fragment of the MAP-1 heavy chain that binds to microtubules and is precipitable with the 34-kD light chain antibody, suggesting that the 34-kD light chain also binds to this domain of the molecule. Since microtubules that contain the 120-kD fragment lack the long lateral projections characteristic of microtubules with intact MAP-l , the 34-kD light chains may be localized at or near the microtubule surface. M ICROTUBULES isolated from brain tissue extracts are composed of tubulin plus several microtubule-associated proteins (MAPs). ~ Of these, two classes of high molecular weight components termed MAP1 and MAP2 have been demonstrated to co-purify with tubulin during cycles of microtubule assembly and disassembly and to stimulate microtubule assembly in vitro (for a review see reference 22). There is considerable evidence that MAPs may mediate the binding of membranous organelles, actin filaments, and intermediate filaments to microtubules, leading to the speculation that they may therefore be important for cellular processes such as mitosis and organelle transport, and for determining the dynamic properties of the cytoskeleton. Immunofluorescence microscopic studies on the distribution of the brain high molecular weight MAPs have shown that MAP-2 is localized primarily in the dendrites of neurons in brain (2, 12) and possibly in small amounts in other cells (6, 17, 23), most notably chromatophores (18). In contrast, MAP-1 is more generally distributed, being found in both dendrites and axons of neurons and in glial cells in brain, in chromatophores, and on both interphase and mitotic microtubules in various tissue culture cells, suggesting that MAP-1 may have a more general function (1, 18). Recently MAP-I has been isolated and demonstrated to stimulate tubulin assembly in vitro (7, 8, 21) and to exhibit structural characteristics similar to those of MAP-2 as 20-nm Abbreviations used in this paper." LC1, LC-2, light chains 1 and 2 of MAP1; MAP, microtubule-associated protein; PMSF, phenylmethylsulfonyl fluoride. long projections on microtubule surfaces (9, 21). Purified preparations of MAP-I from bovine brain have also been demonstrated to contain at least two low molecular weight components that remain tightly associated with MAP1 heavy chains during ion-exchange chromatography and during gel filtration chromatography under nondenaturing conditions. The stoichiometric ratio of both light chains to one MAP1 heavy chain is approximately 1:1 (21 ). In this paper we examine further the relationship of the low molecular weight components to MAP-1 heavy chain using a monoclonal antibody to the larger of the two light chain components and describe the location of this polypeptide on a microtubule-binding, chymotryptic peptide of the MAP-l heavy chain at or near the microtubule surface. Materials and Methods Biochemical Procedures Microtubules, MAPs, MAP-I, and tubulin were purified from bovine brain in a buffer containing 50 mM imidazole-HCl (pH 6.7), 50 mM KC1, 0.5 mM MgCI2, 0.1 mM EDTA, 1 mM GTP, 1 mM 2-mercaptoethanol (buffer A) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Unfractionated microtubule proteins were prepared by two cycles of assembly-disassembly (16) as modified by Rodionov et al. (14). Tubulin and MAPs were separated by chromatography on phosphocellulose in buffer A with 1 mM PMSF (25). MAP-I was purified as described in reference 7. Purified MAP-I was often contaminated with polypeptides of lower molecular weight, including polypeptides that co-migrated with MAP-2 during SDS PAGE (see, for example, Fig. 1, lane C and Fig. 6A, 0 rain). These polypeptides accumulated in preparations during storage, suggesting that they are products of MAP-l proteolysis. Im© The Rockefeller University Press, 0021-9525/86/03/1060/07 $1.0