scholarly journals Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated

1997 ◽  
Vol 110 (4) ◽  
pp. 431-437 ◽  
Author(s):  
A. Schneider ◽  
U. Plessmann ◽  
K. Weber
Keyword(s):  
Trypanosoma Brucei ◽  
Posttranslational Modifications ◽  
Protein Biosynthesis ◽  
Mammalian Brain ◽  
Side Chain ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Isopeptide Bond ◽  
Carboxy Terminal ◽  
Trace Components

To determine the spectrum of tubulin variants in cytoskeletons of Trypanosoma brucei carboxy-terminal fragments of alpha- and beta-tubulin were isolated and characterized by sequencing and mass spectrometry. All variants arise by posttranslational modifications. We confirm the presence of tyrosinated and detyrosinated alpha-tubulin. Unexpectedly, but in line with its sequence, beta-tubulin also occurs with and without its carboxy-terminal tyrosine. Both tyrosinated and detyrosinated alpha- and beta-tubulins are extensively glutamylated. Unglutamylated tubulins are only trace components of the cytoskeletal microtubules. The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for alpha- and beta-tubulin, respectively. The oligoglutamyl side chain is linked via an isopeptide bond to glutamic acid residues 445 of alpha- and 435 of beta-tubulin. The same sites are used in glutamylated tubulins of mammalian brain. No tubulin variants based on polyglycylation are detected in cytoskeletal preparations or in isolated flagella. Tubulin specific incorporation of radioactive glutamate but not of glycine is observed when protein biosynthesis is completely inhibited in Trypanosoma cells. Possible reasons for the absence of polyglycylated tubulins from the trypanosomal axoneme are discussed. Finally we show that lysine 40 of the flagellar alpha-tubulin is completely acetylated.

Tubulin folding is altered by mutations in a putative GTP binding motif

1996 ◽  
Vol 109 (6) ◽  
pp. 1471-1478 ◽  
Author(s):  
J.C. Zabala ◽  
A. Fontalba ◽  
J. Avila
Keyword(s):  
Intramolecular Interaction ◽  
Terminal Region ◽  
Binding Motif ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Proposed Model ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hydrolysis Of

Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An alpha-tubulin point mutant with a T150-->G substitution (the corresponding residue present in beta-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four beta-tubulin point mutants, including the G148-->T substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (intermediates in the process of tubulin folding). Three other mutants within this region (which approximately encompasses residues 137–152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.

scholarly journals Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme

1987 ◽  
Vol 104 (4) ◽  
pp. 1059-1067 ◽  
Author(s):  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Gradient Centrifugation ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Alpha Beta ◽  
Tubulin Tyrosine Ligase ◽  
Carboxy Terminal

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.

scholarly journals Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells.

1988 ◽  
Vol 106 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cell Types ◽  
Beta Tubulin ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
A Cell ◽  
Complex Regulation ◽  
Carboxy Terminal ◽  
Different Cell Types

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families.

scholarly journals Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

1989 ◽  
Vol 9 (10) ◽  
pp. 4291-4297 ◽  
Author(s):  
E Patzelt ◽  
K L Perry ◽  
N Agabian
Keyword(s):  
Trypanosoma Brucei ◽  
Branch Point ◽  
Splice Site ◽  
Mammalian Cells ◽  
Branch Points ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Phosphodiester Bond ◽  
Trans Splicing ◽  
Covalently Linked

The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes.

scholarly journals Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins

1995 ◽  
Vol 108 (1) ◽  
pp. 357-367 ◽  
Author(s):  
Y. Saoudi ◽  
I. Paintrand ◽  
L. Multigner ◽  
D. Job
Keyword(s):  
Tau Proteins ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Microtubule Associated Proteins ◽  
Microtubule Stabilization ◽  
Microtubule Stability ◽  
Associated Proteins ◽  
Carboxy Terminal ◽  
Normal Sensitivity ◽  
Marked Resistance

The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.

Isolation of tubulin polyglutamylase from Crithidia; binding to microtubules and tubulin, and glutamylation of mammalian brain alpha- and beta-tubulins

1999 ◽  
Vol 112 (13) ◽  
pp. 2185-2193 ◽  
Author(s):  
S. Westermann ◽  
A. Schneider ◽  
E.K. Horn ◽  
K. Weber
Keyword(s):  
Glutamic Acid ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Mammalian Brain ◽  
Side Chain ◽  
Dependent Manner ◽  
Sds Page ◽  
Microtubular Cytoskeleton ◽  
Low Efficiency ◽  
Carboxy Terminal

Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzymatic activity that incorporates radioactive glutamic acid in a Mg2+-ATP-dependent manner into alpha- and beta-tubulins. The tubulin polyglutamylase is extracted by 0.25 M salt. The Crithidia enzyme can be purified by ATP-affinity chromatography, glycerol-gradient centrifugation and ion-exchange chromatography. After extraction from the microtubular cytoskeleton the glutamylase forms a complex with alphabeta tubulin, but behaves after removal of tubulin as a globular protein with a molecular mass of 38x10(3). In highly enriched fractions a corresponding band is the major polypeptide visible in SDS-PAGE. The enzyme from Crithidia recognises mammalian brain tubulin, where it incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins. Synthetic peptides with an oligoglutamyl side chain, corresponding to the carboxy-terminal end of brain alpha- and beta-tubulins, are accepted by the enzyme, albeit at low efficiency. The polyglutamylase elongates the side chain by up to 3 and 5 residues, respectively. Other properties of the tubulin polyglutamylase are also discussed.

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

scholarly journals Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains.

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428 ◽  
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

scholarly journals Five mouse tubulin isotypes and their regulated expression during development.

1985 ◽  
Vol 101 (3) ◽  
pp. 852-861 ◽  
Author(s):  
S A Lewis ◽  
M G Lee ◽  
N J Cowan
Keyword(s):  
Developmental Expression ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Genes ◽  
Mouse Tissues ◽  
Tubulin Isotype ◽  
Specific Mrnas ◽  
Beta 2 ◽  
Carboxy Terminal

We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M alpha 2) that encode alpha-tubulin and three (M beta 2, M beta 4, and M beta 5) that encode beta-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two alpha-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the beta-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three beta-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse beta-tubulin isotype and a chicken beta-tubulin isotype. The significance of the interspecies conservation of tubulin isotypes is discussed in relationship to microtubule function. We have used non-cross-hybridizing 3' noncoding region probes from the five cloned mouse tubulin cDNAs to study the developmental expression of each isotype in various mouse tissues. M alpha 1 and M beta 2 are expressed in an approximately coordinate fashion, and their transcripts are most abundant in brain and lung. M alpha 2 and M beta 5 are ubiquitously expressed and to a similar extent in each tissue, with the greatest abundance in spleen, thymus, and immature brain. In contrast, M beta 4 is expressed exclusively in brain. Whereas the expression of the latter isotype increases dramatically during postnatal development, transcripts from all four other tubulin genes decline from maximum levels at or before birth. Tissue-specific development changes in the abundance of tubulin isotype-specific mRNAs are discussed in relationship to organogenesis in the mouse.

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei

1989 ◽  
Vol 9 (10) ◽  
pp. 4291-4297
Author(s):  
E Patzelt ◽  
K L Perry ◽  
N Agabian
Keyword(s):  
Trypanosoma Brucei ◽  
Branch Point ◽  
Splice Site ◽  
Mammalian Cells ◽  
Branch Points ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Phosphodiester Bond ◽  
Trans Splicing ◽  
Covalently Linked

The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes.

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