N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin

Watakabe, A., Kobayashi, R., Helfman, D. M. (1996) N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin. Journal of Cell Science, 109 (Pt 9). pp. 2299-310. ISSN 0021-9533

URL: http://www.ncbi.nlm.nih.gov/pubmed/8886980

Abstract

We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.

Item Type: Paper
Uncontrolled Keywords: Amino Acid Sequence Animals Base Sequence Brain/ metabolism Carrier Proteins/genetics/isolation & purification/ metabolism Cells, Cultured Cloning, Molecular DNA Primers/genetics DNA, Complementary/genetics Female Fetus/metabolism Microfilament Proteins Molecular Sequence Data Neurons/metabolism Pregnancy Protein Binding Rats Recombinant Proteins/genetics/isolation & purification/metabolism Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Sequence Homology, Amino Acid Tropomodulin Tropomyosin/ metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
organs, tissues, organelles, cell types and functions > organs types and functions > brain
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > cDNA
Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning

organs, tissues, organelles, cell types and functions > organs types and functions > metabolism
CSHL Authors:
Communities: CSHL labs > Helfman lab
CSHL labs > Kobayashi lab
Depositing User: Kathleen Darby
Date Deposited: 12 May 2014 16:48
Last Modified: 12 May 2014 16:48
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30122

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