The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform

Martin A Gorovsky - One of the best experts on this subject based on the ideXlab platform.

  • acetylation of lysine 40 in Alpha Tubulin is not essential in tetrahymena thermophila
    Journal of Cell Biology, 1995
    Co-Authors: Jacek Gaertig, Manuel A Cruz, Josephine Bowen, David G Pennock, Long Gu, Martin A Gorovsky
    Abstract:

    In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of Alpha- and beta-Tubulin heterodimer, precluding distinctions among microtubular systems based on Tubulin primary sequence isotypes. Tetrahymena Tubulins also are modified by several types of posttranslational reactions including acetylation of Alpha-Tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal Alpha-Tubulin and numerous other microtubules are acetylated. We completely replaced the single type of Alpha-Tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated Tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated Tubulin are indistinguishable from wild-type cells. Thus, acetylation of Alpha-Tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal Tubulin from cells unable to acetylate Alpha-Tubulin leads us to conclude that: (a) most or all ciliary Alpha-Tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated Alpha-Tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.

  • Regulation and evolution of the single Alpha-Tubulin gene of the ciliate Tetrahymena thermophila
    Cell motility and the cytoskeleton, 1994
    Co-Authors: Kathleen E. Mcgrath, Daniel P. Heruth, Anne A. Kelly, Martin A Gorovsky
    Abstract:

    The single Alpha-Tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other Alpha-Tubulin sequences revealed that it belongs to a group of more evolutionarily constrained Alpha-Tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal Alpha-Tubulins. The single Alpha-Tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved Alpha-Tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal Tubulins are consistent with their simpler microtubule systems. The animal variant Alpha-Tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized Tubulins. To analyze the role of Tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, Alpha-Tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, Alpha-Tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts Alpha-Tubulin message amounts via message decay. However the complex control of Alpha-Tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley-Liss, Inc.

  • Drugs affecting microtubule dynamics increase Alpha-Tubulin mRNA accumulation via transcription in Tetrahymena thermophila.
    Molecular and cellular biology, 1992
    Co-Authors: L A Stargell, Jacek Gaertig, Daniel P. Heruth, Martin A Gorovsky
    Abstract:

    In cultured mammalian cells, an increase in the amount of Tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in Tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of Tubulin message with no change in transcriptional activity of Tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of Tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in Alpha-Tubulin synthesis and Alpha-Tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of Alpha-Tubulin sequences measured by run-on transcription, while the half-life of Tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in Alpha-Tubulin synthesis via an increase in message abundance due to an increase in transcription of the Alpha-Tubulin gene. These results indicate that Tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.

Bernard Eddé - One of the best experts on this subject based on the ideXlab platform.

  • Glutamylation on Alpha-Tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.
    Eukaryotic Cell, 2008
    Co-Authors: Dorota Wloga, Bernard Eddé, Virginie Redeker, Krzysztof Rogowski, Neeraj Sharma, Juliette Van Dijk, Carsten Janke, Marie-hélène Bré, Nicolette Levilliers, Jianming Duan
    Abstract:

    Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated Tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the Alpha-Tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of Alpha-Tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved Tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as Alpha-Tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on Alpha-Tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

  • The polyglutamylated lateral chain of Alpha-Tubulin plays a key role in flagellar motility.
    Journal of cell science, 1996
    Co-Authors: C Gagnon, Bernard Eddé, E. Desbruyeres, D White, J Cosson, P Huitorel, L Paturle-lafanechère, L Multigner, D Job, C Cibert
    Abstract:

    To investigate whether a specific isotype of Tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antiTubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly Alpha-Tubulin in sea urchin sperm axonemes and equally well brain Alpha- and beta-Tubulins. Subtilisin cleavage of Tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of Alpha-Tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated Alpha-Tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of Alpha-Tubulin. On the other hand, other Tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of Alpha-Tubulin plays a dynamic role in a dynein-based motility process.

  • Polyglutamylated Alpha-Tubulin can enter the tyrosination/detyrosination cycle.
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

  • polyglutamylated Alpha Tubulin can enter the tyrosination detyrosination cycle
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Philippe Denoulet - One of the best experts on this subject based on the ideXlab platform.

  • Polyglutamylated Alpha-Tubulin can enter the tyrosination/detyrosination cycle.
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

  • polyglutamylated Alpha Tubulin can enter the tyrosination detyrosination cycle
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Jean Rossier - One of the best experts on this subject based on the ideXlab platform.

  • Posttranslational modifications of the C-terminus of Alpha-Tubulin in adult rat brain: Alpha 4 is glutamylated at two residues.
    Biochemistry, 1998
    Co-Authors: Virginie Redeker, Jean Rossier, Anthony Frankfurter
    Abstract:

    In adult mammalian brain, the C-terminus of Alpha-Tubulin exhibits a high degree of polymorphism due to a combination of four covalent posttranslational modifications: glutamylation, tyrosination, detyrosination, and removal of the penultimate glutamate residue (C-terminal deglutamylation). Glutamylation is the most abundant. To characterize the glutamylation of Alpha-Tubulin and its relationship with the other modifications, we developed a chromatographic procedure for purifying Alpha-Tubulin C-terminal peptides. The purified peptides were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. In this report, we provide a complete description of the glutamylation of tyrosinated, detyrosinated, and C-terminal deglutamylated isoforms of both Alpha-Tubulin isotypes (Alpha1/2 and Alpha4) expressed in adult rat brain. In particular, we describe for the first time the glutamylation of Alpha4. More than 90% of the Alpha-Tubulin is glutamylated, and more than 75% of it is nontyrosinated. Alpha4 is more extensively glutamylated than Alpha1/2, containing as many as 11 posttranslationally added glutamate residues. The most abundant Alpha4 isoform is nontyrosinated, containing five posttranslationally added glutamates, whereas the most abundant Alpha1/2 isoforms are nontyrosinated, with only one or two posttranslationally added glutamates. In contrast to Alpha1/2, Alpha4 is glutamylated at two separate residues (Glu-443 and Glu-445) in the sequence 431DYEEVGIDSYEDEDEGEE448. This is the first evidence that glutamylation can occur on two different residues in the same mammalian Tubulin isotype.

  • Polyglutamylated Alpha-Tubulin can enter the tyrosination/detyrosination cycle.
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

  • polyglutamylated Alpha Tubulin can enter the tyrosination detyrosination cycle
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

E. Desbruyeres - One of the best experts on this subject based on the ideXlab platform.

  • The polyglutamylated lateral chain of Alpha-Tubulin plays a key role in flagellar motility.
    Journal of cell science, 1996
    Co-Authors: C Gagnon, Bernard Eddé, E. Desbruyeres, D White, J Cosson, P Huitorel, L Paturle-lafanechère, L Multigner, D Job, C Cibert
    Abstract:

    To investigate whether a specific isotype of Tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antiTubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly Alpha-Tubulin in sea urchin sperm axonemes and equally well brain Alpha- and beta-Tubulins. Subtilisin cleavage of Tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of Alpha-Tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated Alpha-Tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of Alpha-Tubulin. On the other hand, other Tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of Alpha-Tubulin plays a dynamic role in a dynein-based motility process.

  • Polyglutamylated Alpha-Tubulin can enter the tyrosination/detyrosination cycle.
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

  • polyglutamylated Alpha Tubulin can enter the tyrosination detyrosination cycle
    Biochemistry, 1992
    Co-Authors: Bernard Eddé, Jean Rossier, Jean Pierre Le Caer, Jean-claude Promé, E. Desbruyeres, François Gros, Philippe Denoulet
    Abstract:

    We have previously identified a major modification of neuronal Alpha-Tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated Alpha-Tubulin [Edde, B., Rossier, J., Le Caer, J.P., Desbruyeres, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated Alpha-Tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six Alpha-Tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of Alpha-Tubulin, the structure of this region was investigated. [3H]tyrosinated Tubulin was mixed with a large excess of unlabeled mouse brain Tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of Alpha-Tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated Alpha-Tubulin. A quantitative analysis showed that the different tyrosinated forms of Alpha-Tubulin represent a minor (13%) fraction of the total Alpha-Tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)