Cooperative binding of tropomyosin to muscle and Acanthamoeba actin.

Yang, Y Z; Korn, Edward D.; Eisenberg, Evan

doi:10.1016/s0021-9258(18)50295-7

Cited by 78 publications

(16 citation statements)

References 20 publications

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“…When purified tropomyosin was mixed with actin at a low salt concentration (30 mM KC1) in the absence of divalent cations, none of tropomyosin isoforms was able to bind to F-actin (Fig. 5, lanes A and B), as was expected for skeletal muscle tropomyosin (9,(55)(56)(57). However, in the presence of 5 mM Mg ++, ~95 and 100% binding of tropomyosins 1 and 2 (estimated from the staining intensity of tropomyosin in supernatant and pellet on the gel), respectively, were observed, whereas the binding of tropomyosin 3 to F-actin reached only ~75% (Fig.…”

Section: Characterization Of Cef Tropomyosin Isoformsmentioning

confidence: 60%

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Lin¹,

Helfman²,

Hughes³

et al. 1985

The Journal of Cell Biology

124

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Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

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Section: Characterization Of Cef Tropomyosin Isoformsmentioning

confidence: 60%

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Lin¹,

Helfman²,

Hughes³

et al. 1985

The Journal of Cell Biology

124

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“…Erythrocyte tropomyosin exists as homodimers of Mr 29,000 and 27,000 polypeptides, based on oxidative crosslinking experiments (Fowler, V. M., unpublished data), and the Mr 27,000 tropomyosin has a higher apparent affinity and lower Mg :÷ requirement for binding to F-actin than does the Mr 29,000 tropomyosin (20). The actin-binding properties of the Mr 27,000 isoform more closely resemble those of muscle tropomyosin (64), and binding of erythrocyte tropomyosin to F-actin is highly cooperative (Fig. 7 b, and see reference 20), as is muscle tropomyosin (58,64), consistent with the possibility that erythrocyte tropomyosin self-associates along the actin filament.…”

Section: Mechanism Of Tropomodulin Inhibition Of Tropomyosin Binding To F-actinmentioning

confidence: 99%

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin.

Fowler

1990

The Journal of Cell Biology

140

149

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Abstract. Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98 % homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 + 2.4 nm [SD] in diameter) binds to one of the ends of the rodlike tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a noncompetitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7/~M). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in nonmuscle cells and tissues.T ROPOMYOSINS in nonmuscle ceils are thought to influence actin filament assembly and organization and to play a regulatory role in defining domains along actin filaments, based on in vitro biochemical experiments and on immunofluorescence localization in cells. Tropomyosin blocks the interaction of cross-linking proteins such as filamin or a-actinin with actin filaments (47, 52, 65) and tropomyosin-coated actin filaments are resistant to bundling by villin (5), severing by gelsolin (16, 30, 52), and disassembly by actin-depolymerizing proteins (2, 46). Tropomyosin and t~-actinin are localized in a periodic, alternating fashion along actin filament bundles (stress fibers) in cultured cells (33,35,54) and tropomyosin is absent from actin filaments associated with cell junctions, attachment plaques and the foci of polygonal networks in spreading ceils with which tx-actinin is associated (26,34). Similarly, villin is associated exclusively with the actin filament bundles in the microvilli of the intestin...

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“…It is known that most Tpm isoforms bind cooperatively to F-actin ( 74 , 75 , 76 ). Here, the isoforms that bound F-actin with lower affinity displayed higher cooperativity, and this was true among different isoforms as well as among variants of the same isoform expressed in different cell types.…”

Section: Resultsmentioning

confidence: 99%

Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

Carman

Barrie

Domínguez

2021

Journal of Biological Chemistry

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Cooperative binding of tropomyosin to muscle and Acanthamoeba actin.

Cited by 78 publications

References 20 publications

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin.

Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

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