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PMID: 8408219 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.

The Journal of cell biology ·Vol. 123 ·No. 2 ·1993-10-00 ·Pages 357-71

Masson D, Kreis TE

Abstract

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.

MeSH Terms
Alanine/analysis Amino Acid Sequence Amino Acids/analysis Animals Antibodies, Monoclonal Base Sequence Cell Line Cloning, Molecular DNA/analysis,genetics Dogs Epithelial Cells Epithelium/chemistry,ultrastructure Fibroblasts/cytology Fluorescent Antibody Technique Humans Immunoblotting Kidney/cytology Microscopy, Electron Microtubule-Associated Proteins/analysis,genetics Microtubules/chemistry,ultrastructure Molecular Sequence Data Nocodazole/pharmacology Precipitin Tests Proline/analysis Sequence Analysis, DNA Transfection Vero Cells
Chemicals
Amino Acids Antibodies, Monoclonal Microtubule-Associated Proteins microtubule associated protein 7 DNA Proline Alanine Nocodazole
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Masson D
European Molecular Biology Laboratory, Heidelberg, Germany.
Kreis T E
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.123.2.357
Published
1993-10-00
Pages
357-71
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2119845
Subset
IM
Databases
GENBANK
X73882
External Links
PubMed source DOI: 10.1083/jcb.123.2.357 PMC Full Text View journal details
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