Preparation and properties of pure tubulin S

Knipling, Leslie; Hwang, Jennifer; Wolff, J.

doi:10.1002/(sici)1097-0169(1999)43:1<63::aid-cm7>3.0.co;2-z

Cited by 35 publications

(29 citation statements)

References 35 publications

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“…This model agrees with the observation that the removal of CTTs by proteolysis with subtilisin increases the resistance of microtubules to depolymerization by high salt and cold (4,41), although other studies disagree with this conclusion (25,42). CTTs are highly negatively charged and could interact with the positively charged surface of the tubulin dimer (35).…”

Section: Discussionsupporting

confidence: 89%

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

et al. 2010

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In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of ␣-and ␤-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (51). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the ␥-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of ␣-and ␤-tubulin (14, 36). Glutamylated microtubules are abundant in projections of neurons (14), axonemes (8,15,17), and centrioles/basal bodies (5, 31) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (1, 5). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (22,50,53). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (20). A loss of PGs1, a protein associated with TTLL1 E-ligase (22, 37), disorganizes sperm axonemes in the mouse (11), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (33). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubuleassociated proteins (MAPs) (6,7,19,27).The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (8, 53). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (53). In Tetrahymena, Ttll6Ap is a ␤-tubulin-preferring E-ligase (22), wi...

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Section: Discussionsupporting

confidence: 89%

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

et al. 2010

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“…1 and 2). [38][39][40][41][42] These substrates include MTs lacking either the β-tubulin C-termini (αβ s MTs) or lacking both α-and β-tubulin C-termini (α s β s MTs)-referred to collectively as SMTs, which allowed us to probe the differences between the tubulin C-termini in MCAK function on MTs (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…[38][39][40][41][42] We were unable to find any spiral tubulin structures when we depolymerized GMPCPP-stabilized α s β s MTs in the absence of MCAK nor could we recreate tubulin spiral structures by adding MCAK to previously depolymerized GMPCPP-stabilized α s β s MTs (data not shown), indicating that the structures that we describe are formed specifically by the depolymerization of α s β s MTs by MCAK.…”

Section: Resultsmentioning

confidence: 99%

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

Hertzer

Walczak²

2008

Cell Cycle

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“…To get further insight into the molecular mechanism by which D1 interferes with microtubule assembly, we determined the structure of the tubulin-D1 complex (Tub-D1) by X-ray crystallography. Tubulin was cleaved by subtilisin (22), which removes C-terminal peptides of both subunits, which are not seen in any of the structures that have been determined (12,14), and does not change the rest of the tubulin structure in any other respect (10). Removal of these heterogeneous C-terminal peptides has allowed us to obtain crystals that diffracted to 2.2 Å resolution.…”

Section: D1 Binds To Curved Gtp-tubulin At Its β-Subunit Longitudinalmentioning

confidence: 99%

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end

Pecqueur

Duellberg²,

Dreier

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

144

152

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Microtubules are cytoskeleton filaments consisting of αβ-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the β-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

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Preparation and properties of pure tubulin S

Cited by 35 publications

References 35 publications

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end

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