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.
REFERENCES
Babcock
G. G.
, Fowler
V. M.
(1994
). Isoform-specific interaction of tropomodulin with skeletal muscle and erythrocyte tropomyosins.
J. Biol. Chem
269
, 27510
–27518
Balasubramanian
M. K.
, Helfman
D. M.
, Hemmingsen
S. M.
(1992
). A new tropomyosin essential for cytokinesis in the fission yeast S. pombe.
Nature
360
, 84
–87
Drees
B.
, Brown
C.
, Barrell
B. G.
, Bretscher
A.
(1995
). Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.
J. Cell Biol
128
, 383
–392
Erdelyi
M.
, Michon
A. M.
, Guichet
A.
, Glotzer
J. B.
, Ephrussi
A.
(1995
). Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization.
Nature
377
, 524
–527
Farah
C. S.
, Reinach
F. C.
(1995
). The troponin complex and regulation of muscle contraction.
FASEB J
9
, 755
–767
Fowler
V. M.
(1987
). Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes.
J. Biol. Chem
262
, 12792
–12800
Fowler
V. M.
(1990
). Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin.
J. Cell Biol
111
, 471
–481
Fowler
V. M.
, Sussmann
M. A.
, Miller
P. G.
, Flucher
B. E.
, Daniels
M. P.
(1993
). Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle.
J. Cell Biol
120
, 411
–420
Gimona
M.
, Watakabe
A.
, Helfman
D. M.
(1995
). Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.
Proc. Nat. Acad. Sci. USA
92
, 9776
–9780
Gregorio
C. C.
, Weber
A.
, Bondad
M.
, Pennise
C. R.
, Fowler
V. M.
(1995
). Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes.
Nature
376
, 83
–86
Had
L.
, Faivre
S. C.
, Legrand
C.
, Mery
J.
, Brugidou
J.
, Rabie
A.
(1994
). Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions.
J. Cell Sci
107
, 2961
–2973
Hegmann
T. E.
, Lin
J. L.
, Lin
J. J.
(1989
). Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies.
J. Cell Biol
109
, 1141
–1152
Ito
M.
, Swanson
B.
, Sussman
M. A.
, Kedes
L.
, Lyons
G.
(1995
). Cloning of tropomodulin cDNA and localization of gene transcripts during mouse embryogenesis.
Dev. Biol
167
, 317
–328
Kira
M.
, Tanaka
J.
, Sobue
K.
(1995
). Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones.
J. Neurosci. Res
40
, 294
–305
Kriajevska
M. V.
, Cardenas
M. N.
, Grigorian
M. S.
, Amba rtsumian
N. S.
, Georgiev
G. P.
, Lukanidin
E. M.
(1994
). Non-muscle myosin heavy chain as a possible target for protein encoded by metastasis-related mts-1 gene.
J. Biol. Chem
269
, 19679
–19682
Lees-Miller
J. P.
, Goodwin
L. O.
, Helfman
D. M.
(1990
). Three novel brain tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene through the use of alternative promoters and alternative RNA processing.
Mol. Cell Biol
10
, 1729
–1742
Liu
H.
, Bretscher
A.
(1992
). Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.
J. Cell Biol
118
, 285
–299
Matsumura
F.
, Yamashiro
S.
(1993
). Caldesmon.
Curr. Opin. Cell Biol
5
, 70
–76
Pittenger
M. F.
, Helfman
D. M.
(1992
). In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.
J. Cell Biol
118
, 841
–858
Pittenger
M. F.
, Kazzaz
J. A.
, Helfman
D. M.
(1994
). Functional properties of non-muscle tropomyosin isoforms.
Curr. Opin. Cell Biol
6
, 96
–104
Prasad
G. L.
, Fuldner
R. A.
, Cooper
H. L.
(1993
). Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene.
Proc. Nat. Acad. Sci. USA
90
, 7039
–7043
Shirao
T.
(1995
). The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: a review.
J. Biochem. (Tokyo)
117
, 231
–236
Stamm
S.
, Casper
D.
, Lees
M. J.
, Helfman
D. M.
(1993
). Brain-specific tropomyosins TMBr-1 and TMBr-3 have distinct patterns of expression during development and in adult brain.
Proc. Nat. Acad. Sci. USA
90
, 9857
–9861
Sueyoshi
T.
, Kobayashi
R.
, Nishio
K.
, Aida
K.
, Moore
R.
, Wada
T.
, Handa
H.
, Negishi
M.
(1995
). A nuclear factor (NF2d9) that binds to the male-specific P450 (cyp 2d-9) gene in mouse liver.
Mol. Cell Biol
15
, 4158
–4166
Sung
L. A.
, Fowler
V. M.
, Lambert
K.
, Sussman
M. A.
, Karr
D.
, Chien
S.
(1992
). Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.
J. Biol. Chem
267
, 2616
–2621
Sung
L. A.
, Lin
J. J.
(1994
). Erythrocyte tropomodulin binds to the N terminus of hTM5, a tropomyosin isoform encoded by the gamma-tropomyosin gene.
Biochem. Biophys. Res. Commun
201
, 627
–634
Sussman
M. A.
, Fowler
V. M.
(1992
). Tropomodulin binding to tropomyosins: Isoform-specific differences in affinity and stoichiometry.
Eur. J. Biochem
205
, 355
–362
Sussman
M. A.
, Sakhi
S.
, Barrientos
P.
, Ito
M.
, Kedes
L.
(1994
). Tropomodulin in rat cardiac muscle. Localization of protein is independent of messenger RNA distribution during myofibrillar development.
Circ. Res
75
, 221
–232
Sussman
M. A.
, Sakhi
S.
, Tocco
G.
, Najm
I.
, Baudry
M.
, Kedes
L.
, Schreiber
S. S.
(1994
). Neural tropomodulin: developmental expression and effect of seizure activity.
Brain Res. Dev. Brain Res
80
, 45
–53
Takenaga
K.
, Nakamura
Y.
, Sakiyama
S.
, Hasegawa
Y.
, Sato
K.
, Endo
H.
(1994
). Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin.
J. Cell Biol
124
, 757
–768
Trabelsi
T. H.
, Fattoum
A.
, Represa
A.
, Dessi
F.
, Ben
A. Y.
, der
T. E.
(1995
). Expression of an acidic isoform of calponin in rat brain: western blots on one-or two-dimensional gels and immunolocalization in cultured cells.
Biochem. J
306
, 211
–215
Warren
K. S.
, Lin
J. L.
, McDermott
J. P.
, Lin
J. J.
(1995
). Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis.
J. Cell Biol
129
, 697
–708
Weber
A.
, Pennise
C. R.
, Babcock
G. G.
, Fowler
V. M.
(1994
). Tropomodulin caps the pointed ends of actin filaments.
J. Cell Biol
127
, 1627
–1635
Weinberger
R.
, Schevzov
G.
, Jeffrey
P.
, Gordon
K.
, Hill
M.
, Gunning
P.
(1996
). The molecular composition of neuronal microfilaments is spatially and temporally regulated.
J. Neurosci
16
, 238
–254
Woo
M. K.
, Fowler
V. M.
(1994
). Identification and characterization of tropomodulin and tropomyosin in the adult rat lens.
J. Cell Sci
107
, 1359
–1367
Zhu
J.
, Bilan
P. J.
, Moyers
J. S.
, Antonetti
D. A.
, Kahn
C. R.
(1996
). Rad, a Novel Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin.
Proc. Nat. Acad. Sci. USA
271
, 768
–773
This content is only available via PDF.
© 1996 by Company of Biologists
1996
-PolarityCFP.png?versionId=5150)
