A novel
microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell
cDNA expression library with an anti-serum raised against microtubule-
binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal
antibodies were generated for the further characterization of this MAP. It is different from the microtubule-
binding proteins of similar molecular weights, characterized so far, by its
nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it
E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells,
E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for
E-MAP-115 increases when they polarize and form
blisters. The molecular characterization of
E-MAP-115 reveals that it is a novel
protein with no significant homologies to other known
proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in
proline and
alanine (PAPA region).
E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant
polypeptides in vivo. Overexpression of this domain of
E-MAP-115 by transfection of fibroblasts lacking significant levels of this
protein with its
cDNA renders microtubules stable to
nocodazole. We conclude that
E-MAP-115 is a microtubule-stabilizing
protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.