Régulation de la Dynamique des Microtubules par code Tubuline

Publications de l’équipe

Année de publication : 2008

Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, Bernard Edde, Carsten Janke (2008 Feb 15)

Polyglutamylation is a post-translational modification with a broad range of substrates.

The Journal of biological chemistry : 3915-22 : DOI : 10.1074/jbc.M705813200 En savoir plus
Résumé

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.

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Année de publication : 2007

Juliette van Dijk, Krzysztof Rogowski, Julie Miro, Benjamin Lacroix, Bernard Eddé, Carsten Janke (2007 May 11)

A targeted multienzyme mechanism for selective microtubule polyglutamylation.

Molecular cell : 437-48 : DOI : 10.1016/j.molcel.2007.04.012 En savoir plus
Résumé

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

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Koji Ikegami, Robb L Heier, Midori Taruishi, Hiroshi Takagi, Masahiro Mukai, Shuichi Shimma, Shu Taira, Ken Hatanaka, Nobuhiro Morone, Ikuko Yao, Patrick K Campbell, Shigeki Yuasa, Carsten Janke, Grant R Macgregor, Mitsutoshi Setou (2007 Feb 27)

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.

Proceedings of the National Academy of Sciences of the United States of America : 3213-8 : DOI : 10.1073/pnas.0611547104 En savoir plus
Résumé

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular « traffic sign » for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

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Année de publication : 2005

Carsten Janke, Krzysztof Rogowski, Dorota Wloga, Catherine Regnard, Andrey V Kajava, Jean-Marc Strub, Nevzat Temurak, Juliette van Dijk, Dominique Boucher, Alain van Dorsselaer, Swati Suryavanshi, Jacek Gaertig, Bernard Eddé (2005 Jun 17)

Tubulin polyglutamylase enzymes are members of the TTL domain protein family.

Science (New York, N.Y.) : 1758-62 : DOI : 10.1126/science.1113010 En savoir plus
Résumé

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

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Année de publication : 2004

Carsten Janke, Maria M Magiera, Nicole Rathfelder, Christof Taxis, Simone Reber, Hiromi Maekawa, Alexandra Moreno-Borchart, Georg Doenges, Etienne Schwob, Elmar Schiebel, Michael Knop (2004 Aug 5)

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Yeast (Chichester, England) : 947-62 : DOI : 10.1002/yea.1142 En savoir plus
Résumé

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

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Année de publication : 2003

Catherine Regnard, Didier Fesquet, Carsten Janke, Dominique Boucher, Elisabeth Desbruyéres, Annette Koulakoff, Christine Insina, Pierre Travo, Bernard Eddé (2003 Oct 15)

Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.

Journal of cell science : 4181-90 : DOI : 10.1242/jcs.00743 En savoir plus
Résumé

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360×103) displayed at least three major polypeptides of 32, 50 and 80×103, present in stochiometric amounts. We show that the 32×103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

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