Local Unfolding and the Stepwise Loss of the Functional Properties of Tubulin

Sackett, Dan L.; Bhattacharyya, Bhabatarak; Wolff, J.

doi:10.1021/bi00209a019

Cited by 50 publications

(58 citation statements)

References 58 publications

(67 reference statements)

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“…Initial interactions form between cognate binding sites on tau and the microtubules, which in turn lead to stabilizing intramolecular interactions within tau that help guide it into a stable, folded microtubule binding conformation. This model accommodates the observation that tubulin is a highly polymorphic protein that changes conformation readily in response to binding interactions (42). Taken together with the proposed highly flexible and extended nature of tau in solution, these properties are ideally suited for an induced fit mechanism, since they would allow subtle changes in tubulin conformation and larger changes in tau conformation.…”

Section: The First Two Repeats and Their Intervening Inter-repeat Funmentioning

confidence: 55%

Structural and Functional Differences between 3-Repeat and 4-Repeat Tau Isoforms

Goode

Chau

Denis

et al. 2000

Journal of Biological Chemistry

207

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Tau, MAP2, and MAP4 are members of a microtubuleassociated protein (MAP) family that are each expressed as "3-repeat" and "4-repeat" isoforms. These isoforms arise from tightly controlled tissue-specific and/or developmentally regulated alternative splicing of a 31-amino acid long "inter-repeat:repeat module," raising the possibility that different MAP isoforms may possess some distinct functional capabilities. Consistent with this hypothesis, regulatory mutations in the human tau gene that disrupt the normal balance between 3-repeat and 4-repeat tau isoform expression lead to a collection of neurodegenerative diseases known as FTDP-17 (fronto-temporal dementias and Parkinsonism linked to chromosome 17), which are characterized by the formation of pathological tau filaments and neuronal cell death. Unfortunately, very little is known regarding structural and functional differences between the isoforms. In our previous analyses, we focused on 4-repeat tau structure and function. Here, we investigate 3-repeat tau, generating a series of truncations, amino acid substitutions, and internal deletions and examining the functional consequences. 3-Repeat tau possesses a "core microtubule binding domain" composed of its first two repeats and the intervening inter-repeat. This observation is in marked contrast to the widely held notion that tau possesses multiple independent tubulin-binding sites aligned in sequence along the length of the protein.In addition, we observed that the carboxyl-terminal sequences downstream of the repeat region make a strong but indirect contribution to microtubule binding activity in 3-repeat tau, which is in contrast to the negligible effect of these same sequences in 4-repeat tau. Taken together with previous work, these data suggest that 3-repeat and 4-repeat tau assume complex and distinct structures that are regulated differentially, which in turn suggests that they may possess isoform-specific functional capabilities. The relevance of isoform-specific structure and function to normal tau action and the onset of neurodegenerative disease are discussed.

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Section: The First Two Repeats and Their Intervening Inter-repeat Funmentioning

confidence: 55%

Structural and Functional Differences between 3-Repeat and 4-Repeat Tau Isoforms

Goode

Chau

Denis

et al. 2000

Journal of Biological Chemistry

207

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“…Subtilisin-truncated tubulin (S-tubulin) and tubulin free from associated proteins were prepared as described (16,17). Chicken erythrocyte tubulin (E-tubulin) was purified from heparinized chicken blood (Pelfreeze Inc., Rogers, AK) by the procedure of Murphy and Wallis (18).…”

Section: Purification Of Cofactors and Labeled Tubulin-to Generate Pumentioning

confidence: 99%

Tubulin Folding Cofactors as GTPase-activating Proteins

Tian¹,

Bhamidipati

Cowan

et al. 1999

Journal of Biological Chemistry

111

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In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized ␣-and ␤-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. ␣-and ␤-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by ␤-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein ␤-tubulin to hydrolyze its GTP.

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“…First, studies of stepwise denaturation of tubulin demonstrate that the most sensitive property of the molecule is its ability to assemble into microtubules (Sackett et al, 1994). Therefore, if tubulin assembles into microtubules, it can be assumed that these molecules are not denatured.…”

Section: Cell-free Palmitoylation Of Tubulinmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Caron

1997

MBoC

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It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(a,3)-methylenediphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

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Local Unfolding and the Stepwise Loss of the Functional Properties of Tubulin

Cited by 50 publications

References 58 publications

Structural and Functional Differences between 3-Repeat and 4-Repeat Tau Isoforms

Structural and Functional Differences between 3-Repeat and 4-Repeat Tau Isoforms

Tubulin Folding Cofactors as GTPase-activating Proteins

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

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