Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.
ABSTRACT 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 360x103) displayed at least three major polypeptides of 32, 50 and 80x103, present in stochiometric amounts. We show that the 32x103 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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ABSTRACT: Amyotrophic lateral sclerosis is heterogeneous with high variability in the speed of progression even in cases with a defined genetic cause such as superoxide dismutase 1 (SOD1) mutations. We reported that SOD1(G93A) mice on distinct genetic backgrounds (C57 and 129Sv) show consistent phenotypic differences in speed of disease progression and life-span that are not explained by differences in human SOD1 transgene copy number or the burden of mutant SOD1 protein within the nervous system. We aimed to compare the gene expression profiles of motor neurons from these two SOD1(G93A) mouse strains to discover the molecular mechanisms contributing to the distinct phenotypes and to identify factors underlying fast and slow disease progression. Lumbar spinal motor neurons from the two SOD1(G93A) mouse strains were isolated by laser capture microdissection and transcriptome analysis was conducted at four stages of disease. We identified marked differences in the motor neuron transcriptome between the two mice strains at disease onset, with a dramatic reduction of gene expression in the rapidly progressive (129Sv-SOD1(G93A)) compared with the slowly progressing mutant SOD1 mice (C57-SOD1(G93A)) (1276 versus 346; Q-value ≤ 0.01). Gene ontology pathway analysis of the transcriptional profile from 129Sv-SOD1(G93A) mice showed marked downregulation of specific pathways involved in mitochondrial function, as well as predicted deficiencies in protein degradation and axonal transport mechanisms. In contrast, the transcriptional profile from C57-SOD1(G93A) mice with the more benign disease course, revealed strong gene enrichment relating to immune system processes compared with 129Sv-SOD1(G93A) mice. Motor neurons from the more benign mutant strain demonstrated striking complement activation, over-expressing genes normally involved in immune cell function. We validated through immunohistochemistry increased expression of the C3 complement subunit and major histocompatibility complex I within motor neurons. In addition, we demonstrated that motor neurons from the slowly progressing mice activate a series of genes with neuroprotective properties such as angiogenin and the nuclear factor (erythroid-derived 2)-like 2 transcriptional regulator. In contrast, the faster progressing mice show dramatically reduced expression at disease onset of cell pathways involved in neuroprotection. This study highlights a set of key gene and molecular pathway indices of fast or slow disease progression which may prove useful in identifying potential disease modifiers responsible for the heterogeneity of human amyotrophic lateral sclerosis and which may represent valid therapeutic targets for ameliorating the disease course in humans.Brain 09/2013; · 9.92 Impact Factor
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ABSTRACT: Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different tubulin isotypes and through post-translational modifications, such as detyrosination and further cleavage to Δ2-tubulin, acetylation, polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many tubulin post-translational modifications has enabled functional studies demonstrating that these post-translational modifications may regulate microtubule functions through an amazing range of mechanisms.Nature Reviews Molecular Cell Biology 11/2011; 12(12):773-86. · 37.16 Impact Factor
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ABSTRACT: Cellular microtubules are marked by abundant and evolutionarily conserved post-translational modifications that have the potential to tune their functions. This review focuses on the astonishing chemical complexity introduced in the tubulin heterodimer at the post-translational level and summarizes the recent advances in identifying the enzymes responsible for these modifications and deciphering the consequences of tubulin's chemical diversity on the function of molecular motors and microtubule associated proteins.Cytoskeleton 03/2012; 69(7):442-63. · 2.87 Impact Factor
Microtubules (MTs) play fundamental roles in multiple
cellular processes (Hyams and Lloyd, 1993) and there is a
growing list of factors and molecular motors that regulate the
organisation and the spatiotemporal remodelling of the MT
network (Drewes et al., 1998; Kobayashi and Mundel, 1998;
McIntosh et al., 2002).
Tubulin the protein that constitutes MTs is highly
heterogeneous, especially in the C-terminal tail, which is
important for the binding of most microtubule-associated
proteins (MAPs). Each of the two subunits (αand β) is encoded
by a multigene family of 6-8 members in mammals. In addition
to this genetic diversity, seven different types of post-
translational modifications have been evidenced so far, which
further increases the heterogeneity of the protein (Luduena,
1998). This heterogeneity is thought to allow MTs to undergo
specialised functions, as formulated a number of years ago in
‘the multi-tubulin hypothesis’ (Fulton and Simpson, 1976). In
the last few decades, numerous observations have supported
this hypothesis (Edde et al., 1987; Edde et al., 1983; Gard and
Kirschner, 1985; Kreitzer et al., 1999) even though there are
many more hallmarks of tubulin heterogeneity with mysterious
Among tubulin modifications, polyglutamylation (Edde et
al., 1990) and polyglycylation (Redeker et al., 1994) are
very unusual post-translational modifications, consisting of
the sequential addition of glutamate and glycine units,
respectively. The first added unit is linked by an isopeptidic
bond to the γ-carboxyl of a glutamate residue of the main
polypeptidic chain. Addition of the other units occurs through
classic peptide bonds and leads to the formation of
polyglutamyl or polyglycyl chains of various lengths.
Interestingly, polyglutamylation is not restricted to tubulin, it
was recently shown to occur on nucleosome assembly proteins
NAP1 and NAP2 (Regnard et al., 2000), nucleoplasmin (our
unpublished results), and probably on several yet unidentified
Polyglutamylation of tubulin is a widespread modification
prevailing in centrioles (Bobinnec et al., 1998b), basal
bodies and axonemes (Fouquet et al., 1994; Gagnon et al.,
1996). In neurones, most of the tubulin is polyglutamylated
(Audebert et al., 1994) whereas it is present, albeit at low
levels in most, if not all, non-neuronal cells and tissues
analysed (Wolff et al., 1994; Regnard et al., 1999; Kann et
An increasing amount of data strongly suggests a major role
for polyglutamylation in regulating MT functions. Indeed,
masking of the polyglutamyl side chain with a specific
monoclonal antibody (GT335) was shown to strongly inhibit
axonemal beating (Gagnon et al., 1996; Million et al., 1999)
and led to the disappearance of centrioles and the dispersion
of the pericentriolar material in cells (Bobinnec et al., 1998a).
In addition, the extent of the modification seems to have a fine
tuning effect since the in vitro binding of various MAPs and
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.
Supplemental data available online
Key words: Axoneme, Cell cycle, Centrosome, Neurone,
Characterisation of PGs1, a subunit of a protein
complex co-purifying with tubulin polyglutamylase
Catherine Regnard2,*, Didier Fesquet1, Carsten Janke1, Dominique Boucher2, Elisabeth Desbruyères2,
Annette Koulakoff3, Christine Insina1, Pierre Travo1and Bernard Eddé1,‡
1Centre de Recherches de Biochimie Macromoléculaire, CNRS, 34293 Montpellier, France
2Laboratoire de Biochimie Cellulaire, CNRS, Université Paris 6, 75252 Paris, France
3INSERM U114, Collège de France, 75005 Paris, France
*Present address: Department of Molecular Biology, Adolf Butenandt Institute, 80336 Munich, Germany
‡Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 4 July 2003
Journal of Cell Science 116, 4181-4190 © 2003 The Company of Biologists Ltd
MT motors are influenced by the length of the polyglutamyl
side chain (Bonnet et al., 2001; Boucher et al., 1994; Larcher
et al., 1996).
To better understand the function of polyglutamylation,
identification of polyglutamylase(s) is a crucial step. We
believe that mouse brain tubulin polyglutamylase (TPG) is a
multimeric complex composed of at least three different
polypeptides of Mr 32, 50 and 80×103and we describe the
function of the 32×103subunit, called PGs1, as an addressing
subunit for a subset of polyglutamylases, including neuronal,
centriolar and axonemal TPGs.
Materials and Methods
Plasmids and protein expression
Mouse PGs1 cDNA was PCR amplified from mouse brain cDNA and
cloned into pET15b (Novagen) and into pEGFP-N1 (Clonetech).
PGs1 was expressed in E. coli BL21 DE3. Inclusion bodies were
obtained from the pellet of the crude cell extract by several washing
steps in 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA, 5%
Triton X-100. PGs1 accounted for >90% of the proteins of the
∆mPGs1 was amplified from the vector pEGFP-mPGs1 in two
steps: (1) amplification of the 5′ fragment (1-165) with a 5′ primer
and a fusion primer (bridging the deletion); (2) amplification of the
3′ part of ∆mPGs1 (255-912) with the PCR-product of step 1 and a
3′ primer. The PCR-product of step 2 was exchanged with mPGs1 in
pEGFP-mPGs1 to obtain pEGFP-∆mPGs1 (∆166-254). Sequencing
and western blot analysis revealed the expression of the correct mutant
Northern mouse tissue blot (from V. Coulon, IGM, Montpellier,
France) was hybridised with radio-labelled PGs1 cDNA as described
by De Toledo et al. (De Toledo et al., 2001).
Monoclonal antibodies (mAbs) against a 400× purified fraction of
TPG [fraction IV (Regnard et al., 1998)] were generated by injecting
50 µg into 8-week-old BALB/c mice. Cell fusion with mouse
myeloma cells P3-X63-Ag8.653 was carried out (de StGroth and
Scheidegger, 1980). Out of 322 hybridomas, 11 positive clones,
characterised by the ability to deplete TPG activity, were obtained.
mAb206 (IgG2a, κ) was affinity purified on a Sepharose-Protein G
column (Amersham Pharmacia Biotech) from ascite fluid from clone
206 (Eurogentec, Belgium).
Polyclonal antibody L83 was raised in rabbit against PGs1
inclusion bodies and purified by affinity chromatography on PGs1
linked to CNBr-activated Sepharose (Amersham Pharmacia Biotech).
The specificity of L83 was confirmed by western blot analysis.
Other antibodies used in this study were mAb GT335 (Wolff et al.,
1994), mAb anti-γ-tubulin (GTU-88; Sigma), mAb anti-α-tubulin
(DM1A; Amersham Pharmacia Biotech), mAbs anti-RIIα and -RIIβ
of PKA (Transduction Laboratories). Anti-centrosome mAb
(CTR453) was a gift from Dr G. Keryer (Institut Curie, Paris, France).
Its epitope was recently mapped to centrosomal AKAP450 (Keryer et
Tubulin polyglutamylase activity was measured by the incorporation
of L-[3H]glutamate (45-55 Ci/mmol; Amersham, UK) into taxotere-
stabilised MTs, as described previously (Regnard et al., 1998).
Purification and identification of mouse brain TPG
A TPG fraction IV was obtained from 150 3-day-old mouse brains as
described by Regnard et al. (Regnard et al., 1998), and concentrated
by ammonium sulfate precipitation at 40% saturation. All steps were
performed at 4°C. The pellet was solubilised in TBSTx (50mM Tris-
HCl, pH7.6, 100mM NaCl, 0.1% Triton X-100), containing protease
inhibitors, cleared by centrifugation, and pre-incubated with proteinG
magnetic beads (Dynal Biotech) bound to mouse non-specific Igs
(Sigma). The supernatant was incubated overnight with 100 µl bead
suspension bound to affinity-purified mAb206. The beads were
extensively washed with TBSTx; 0.5 M NaCl, and bound proteins
were resolved on a 10% SDS gel and stained with Coomassie Blue
(Serva). Protein bands were excised and digested with trypsin (Seq.
Aliquots of analytes solutions (0.7 µl) were mixed with the same
volume of alpha-cyano-4-hydroxy-trans-cinnamic acid as matrix
(10 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid) and loaded
onto the probe. Analysis was performed using a Biflex III MALDI-
TOF mass spectrometer (Bruker-Franzen Analytik, Bremen,
Germany) in reflectron mode. The accelerating voltage was 20 kV
with a delayed extraction of 400 nseconds. Spectra were analysed
using XTOF software (Bruker-Franzen Analytik). Autoproteolysis
products of trypsin (Ms: 842.51, 1045.56, 2211.10) were used as
internal calibrates. The p32 protein was identified by Mascot search
Electrophoresis and western blotting
1D-PAGE was performed according to the method of Laemmli,
(Laemmli, 1970). Protein concentration was determined with
Bradford reagent (Sigma). Affinity-purified L83 was diluted at
0.2 µg/ml and donkey anti-rabbit Ig (Amersham Pharmacia
Biotech) 1:10,000 in PBSTw (PBS; 0.1% Tween 20). Blots were
developed with Western Lightning Chemo-luminescence Reagent
Cell culture and transfection
Multipotential embryonal carcinoma cell line PCC7-AzaR1 1009
(Pfeiffer et al., 1981), here denoted 1009, was obtained from the
Pasteur Institute (Paris, France). Cells were grown in DME medium
(Invitrogen) containing 15% foetal calf serum. Cells blocked in
mitosis were collected after an 11-hour incubation with 1 µM
nocodazole (Sigma). Cell transfection was carried out at 30%
confluence using Lipofectamine Plus reagent (Invitrogen). Stable
transfected 1009 cells were selected through several passages in
culture medium containing 0.8 mg/ml geneticin (Invitrogen).
Neurones were isolated from foetal mouse brains at 15 days of
gestation and allowed to develop in culture, as described previously
(Berwald-Netter et al., 1981). Neural cells depleted in neurones were
cultured as described previously (Alvarez-Buylla et al., 2001).
1009 cells were cultured on glass coverslips under standard culture
conditions and fixed either with 3.7% paraformaldehyde at room
temperature, for 15minutes or with methanol at –20°C, for 8minutes
and incubated with L83 (2µg/ml), GT335 (5µg/ml), GTU-88 (1:500),
CTR453 (1:10), DM1A (1:1000) or anti-RIIα and β (1:150) for
1 hour, followed by 30 minutes with either donkey anti-rabbit Ig,
Texas Red-conjugated (Amersham Pharmacia Biotech; 1:200), or
Rhodamine- or Fluoresceine-conjugated goat anti-mouse Ig (Jackson
ImmunoResearch Laboratories; 1:200). Coverslips were mounted
with Vectashield mounting medium (Vector Laboratories).
Journal of Cell Science 116 (20)
4183 PGs1, a subunit of tubulin polyglutamylase
Microscopes used were DMIRBO or DMRA microscopes (Leica,
Germany). For confocal microscopy, a DMRA microscope was
equipped with a module Ultraview (Perkin Elmer, UK). Imaging
software used was Metamorph (Universal Imaging Copp., USA). For
deconvolution and 3-D reconstruction, image stacks were processed
on a SGI octane workstation running Huygens (Scientific Volume
Imaging b.v., Netherlands) using MLE algorithms. 3-D restored stacks
were processed with Imaris 3 (Bitplane, Switzerland) for volume
rendering. Live cells were observed with a DM IRBE inverted
microscope with a PL APO 63× oil immersion objective.
Purification of brain TPG
We have previously reported a 1000-fold purification of tubulin
polyglutamylase from 3-day-old mouse brain. This partially
purified fraction still contained a number of protein species,
preventing the identification of the enzyme (Regnard et al.,
1998). We thus decided to develop an alternative strategy and
produced monoclonal antibodies against the 400× purified
fraction of TPG. These antibodies were selected for their
capacity to immuno-precipitate the enzymatic activity from
brain TPG fractions.
Antibodies from each hybridoma were bound to a mixture
of protein A and protein G and incubated with fraction II [a
20× purified fraction (Regnard et al., 1998)]. TPG activity was
then measured in the supernatant and bead fraction (Fig. 1,
upper and lower panels, respectively). Of the 330 tested
hybridomas, 11 were able to deplete TPG, albeit with variable
efficiency (30-90% depletion). In each instance, both α- and
β-TPG activities were depleted to the same extent (β:α ratio,
1:3). TPG activity was recovered in the bead fractions but with
a low yield, presumably because the beads hinder proper
interaction of the bound enzyme with the large MT substrates.
None of the selected hybridomas produced antibodies able to
significantly inhibit TPG activity in vitro or to detect protein
by western blot analysis.
As mAb206 has the best efficiency, it was used for further
purification of TPG by immunoprecipitation of fraction IV
(Fig. 2A). Apart from the antibody heavy (HC) and light (LC)
Fig. 1. Screening of anti-TPG hybridomas. Of the 330 hybridomas
tested, 15 are presented here. TPG activity was measured in both the
supernatant (top) and bead fraction (bottom) of each IP.
Incorporation of [3H]Glu into α-tubulin (light-grey bars) and β-
tubulin (dark-grey bars) was measured separately for the
supernatants and together for the pellets (black bars). The control
(Ctl) is an IP performed with the culture medium alone. Numbering
refers to hybridoma names.
Fig. 2. Immunoprecipitation of brain TPG and identification of the
PGs1 subunit. (A) Silver staining of Fraction IV (lane 1) and the IP
fraction obtained with mAb206 (lane 2). In addition to the light (LC)
and heavy (HC) antibody chains (arrowheads), three protein species
(arrows) are found at high levels in the IP fraction. (B)
Immunoprecipitation was performed with mAb206 and analysed by
western blotting with L83. Comparable amounts of fraction IV (lane
1), unbound fraction (lane 2) and IP fraction (lane 3) were loaded on
the gel. (C) L83 immunoprecipitates TPG activity. S1-S3 correspond
to the IP supernatants obtained after the first, second and third cycle
of IP, and B1-B3 to the bead fractions. Similar proportions of each
fraction were tested for TPG activity (upper panel) and analysed by
western blotting with L83 (lower panel). The position of p32 and of
the antibody light chain are indicated by arrows. (D) Enrichment of
PGs1 during TPG purification. Brain fractions I (initial supernatant)
and II-IV [20×, 160× and 400× purified fractions, respectively, as
described by Regnard et al. (Regnard et al., 1998)] were analysed by
western blotting with L83. Equal amounts of proteins were loaded in
each lane. (E) PGs1 mRNA expression profile. Total RNA from
various mouse tissues were analysed by northern blotting and probed
with a 32P-labelled PGs1 cDNA ORF.
chains, three prominent protein species at Mr 32, 50 and
80×103were detected in the immunoprecipitate by silver
staining of the gel (Fig. 2A). Thus, TPG appears to be a
multimeric complex. A dimer of the three proteins would give
a total mass of 324×103, which is in good agreement with the
previously measured mass (360×103) of the enzyme in native
conditions (Regnard et al., 1998).
Identification of PGs1
Tryptic digests from each protein in the gel were analysed by
mass spectrometry. Database searches identified the 32×103
protein as the product of a mouse gene called GTRGEO22 (in
Mascot search, this was the only sequence which gave a
significant probability based mowse score, 93). This gene is
composed of two small exons separated by a large intron and
is located on chromosome 10 (accession number, AF303106).
It encodes a 303 amino acid protein (Mr 32.6×103, pI 8.95)
of unknown function, containing a putative A-kinase-
anchoring protein (AKAP) binding domain in the first exon
(ProtFam accession number: PF02297). The protein is
conserved in human (locus 19p13.3) and other vertebrates
as well as in the invertebrate Ciona intestinalis (see
Supplementary Fig. 1: http://jsc.biologists.org/supplemental)
but no clear orthologues were found in plants, Drosophila or
We cloned the cDNA corresponding to the mouse
GTRGEO22 gene and raised polyclonal antibodies against
bacterially expressed recombinant protein. Affinity-purified
antibodies (hereafter denoted L83) specifically reacted with
the recombinant protein (data not shown) and with the protein
species of 32×103in fraction IV (Fig. 2B, lane 1). The p32
protein is efficiently immunoprecipitated with mAb206 (Fig.
2B, lanes 2 and 3) along with 90% of TPG activity. Likewise,
L83 immunoprecipitates both p32 protein and TPG activity.
When three consecutive cycles of immunoprecipitation are
performed, ~90% of TPG activity is depleted and recovered
in the consecutive bead fractions (Fig. 2C). To further confirm
that p32 is genuinely part of the TPG complex, the different
fractions obtained during the purification procedure were
analysed by western blotting with L83. As shown in Fig. 2D,
a very faint signal was detected in the starting brain fraction
(fraction I). This signal increased strongly in parallel with the
extent of TPG purification (fractions II-IV) [see Regnard et
al. (Regnard et al., 1998) for a description of the different
fractions]. Therefore, the p32 protein is subsequently called
PGs1 for polyglutamylase subunit 1.
Expression pattern of PGs1
Western blot analysis of PGs1 in a number of cell and tissue
extracts did not yield detectable signals (data not shown),
except in brain (Fig. 2D). However, northern blot analysis on
various mouse tissues shows that PGs1 mRNA is present at
low levels in most adult tissues tested, and at a significantly
higher level in brain (Fig. 2E). These results correlate with
the abundance of polyglutamylated tubulin as well as with the
distribution of TPG activity, which is higher in extracts from
brain and neurones than in other tissues and cell lines
(Regnard et al., 1998; Regnard et al., 1999; Wolff et al.,
PGs1 is associated with major sites of tubulin
We next analysed the subcellular distribution of PGs1 by
immunofluorescence on primary cultures of neurones from
embryonic mouse cortex. In young neurones, after 2 days in
culture, L83 stains several dots in the cell soma, which are
more concentrated in the region where neurites emerge (Fig.
Journal of Cell Science 116 (20)
Fig. 3. Confocal images of PGs1 localisation in mouse neural cells in
primary culture. (A-C) Mouse brain neurones were cultured for 2
(A), 8 (B) and 13 (C) days, then fixed with paraformaldehyde and
double stained with L83 (left panels, green) and GT335 (middle
panels, red). Right panels present the corresponding merged images.
(D,E), Mouse brain neural cells fixed with paraformaldehyde and
double stained with L83 (left panels in D and E, green) and GT335
(middle panel in D, red) or anti-γ-tubulin mAb GTU-88 (middle
panel in E, red). Right panels show the corresponding merged
images. Staining of PGs1 was observed at the base of primary cilia
(arrowheads) as well as of multiple cilia emerging from ependymal
cells (arrows). Note also some PGs1 staining along the cilia. Scale
4185 PGs1, a subunit of tubulin polyglutamylase
3A). In more differentiated neurones, after 8 to 13 days in
culture (Fig. 3B,C), a stronger punctuate labelling is detected
both in the cell somata and in the proximal part of the neurites.
Double staining of the neurones with L83 and GT335, a mAb
raised against the polyglutamylated motif (Wolff et al., 1994)
showed a restricted co-localisation. The majority of PGs1 is in
the proximal part of neurites whereas only some dot-like
structures localise along the entire length of neurites, which
contain the strongly polyglutamylated microtubules (Fig. 3A-
To determine whether PGs1 is a neurone-specific subunit of
TPG or is shared by other polyglutamylases, we analysed its
distribution in other cell types. Ciliated and flagellated cells
are of particular interest since axonemes contain strongly
polyglutamylated tubulins (Fouquet et al., 1994; Gagnon et al.,
1996; Million et al., 1999). Mixed neural cell cultures depleted
in neurones contain, in addition to astrocytes, ependymal cells
characterised by the presence of multiple cilia and cells that
exhibit a single cilium (Alvarez-Buylla et al., 2001). In the
latter, L83 decorates the base of the cilium (Fig. 3D,
arrowhead). Similar but stronger staining is observed at the
base of multiple cilia emerging from ependymal cells (Fig. 3D,
arrows). Some dots are also observed along the length of cilia
indicating a partial co-localisation of PGs1 with the strongly
polyglutamylated MTs of axonemes. Double labelling with
L83 and anti-γ-tubulin antibodies reveal an overlap of PGs1
and γ-tubulin in all cells present in the culture, suggesting that
PGs1 is also present at the centrosomes (Fig. 3E). Finally, in
mouse spermatozoa, a strong concentration of PGs1 in the
proximal part of the flagella is observed (data not shown).
Taken together, these results show that PGs1 is enriched
in the vicinity of major sites of tubulin polyglutamylation,
i.e. neurites, centrosomes, basal bodies and axonemes. There
is, however, no strict co-localisation of PGs1 and
polyglutamylated tubulin since the strongly glutamylated MTs
in neurites and ciliary axonemes are poorly labelled with L83.
PGs1 is addressing TPG to the centrosome in
exponentially growing cells
In exponentially growing cells the overall polyglutamylation of
tubulin is low and restricted to the β-tubulin subunit, except for
the highly modified centriolar MTs, which are mostly
glutamylated on the α-tubulin subunit (Bobinnec et al., 1998b).
To better examine the association of PGs1 with the centrosome
we used the mouse teratocarcinoma cell line 1009, which can
be grown as multipotential embryonal carcinoma (EC) cells or
induced to differentiate into neurone-like cells (Edde et al.,
1983; Lang et al., 1989). In EC cells, PGs1 is detected as a
punctuate stain focusing in one region of the cell (Fig. 4). Co-
labelling with GT335, GTU-88 (anti-γ-tubulin) and CTR-453
(anti-AKAP450) shows that PGs1 staining includes the
centrioles and overlaps with γ-tubulin and AKAP450 staining
Centrosomal localisation of PGs1 is further confirmed by
transient expression of PGs1-GFP in 1009 EC cells. Labelling
with L83 shows a complete co-localisation with the green
fluorescence indicating the specificity of our antibody as well
as the correct localisation of the overexpressed and tagged
protein (Fig. 5A). In all transfected cells, PGs1-GFP
accumulates around the centrosome, as evidenced by labelling
with mAb CTR-453 (Fig. 5B). Control cells transfected with
GFP alone present a diffuse staining in the whole cytoplasm
Stable cell lines expressing PGs1-GFP were established.
The GFP-tagged protein has the proper localisation at the
centrosome both in paraformaldehyde-fixed cells (Fig. 5C) and
in unfixed cells (Fig. 5D). Western blotting with L83 shows
that only PGs1-GFP (~60×103) is detected in total cell extracts
suggesting that the tagged protein is at least in a 10-fold excess
compare to the endogenous PGs1 (Fig. 5E). Thus, the
mechanism enabling PGs1-GFP localisation at the centrosome
is not saturated. In addition, neither the GT335 reactivity nor
the TPG activity is significantly increased in those cells (not
shown), suggesting that PGs1 has no glutamylation activity
on its own. This subunit is most probably implicated in
intracellular addressing of the TPG protein complex.
The putative AKAP binding domain of PGs1 has no role
in centrosomal localisation
PGs1 contains a sequence that exhibits homology to the AKAP-
binding domain of cAMP-dependent protein kinase (PKA;
Supplementary Fig. 1: http://jsc.biologists.org/supplemental).
This domain is found in the RII subunits of PKA and has been
shown to be implicated in targeting the kinase at specific
subcellular sites, one of which is the centrosome, through
interaction with AKAPs (for reviews, see Colledge and Scott,
1999; Diviani and Scott, 2001). We examined the role of this
Fig. 4. PGs1 is localised at the centrosome in 1009 EC cells.
Exponentially growing 1009 EC cells were fixed with methanol and
double stained with L83 (left panels) and anti-polyglutamylated
tubulin (GT335, right panel in A) or anti-γ-tubulin (GTU-88, right
panel in B), or anti-AKAP450 (CTR-453, right panel in C). Scale
sequence in PGs1 targeting. Double staining of PGs1 with
either the RIIα or RIIβ subunit of PKA shows co-localisation
of both proteins at the centrosome (Fig. 6A,B), raising the
possibility that PGs1 could be targeted to the centrosome by the
same mechanism as PKA. However, overexpression of PGs1-
GFP in 1009 cells does not modify the centrosomal localisation
of PKA, indicating that PGs1 and PKA do not compete with
each other for the same sites on the centrosome (Fig.6C,D). In
addition, an overexpressed ∆PGs1-GFP mutant protein that is
missing the putative AKAP-binding motive (amino acids 56-
85), localises at the centrosome in a manner indistinguishable
from that of the wild-type PGs1-GFP fusion protein (not
shown). These results indicate that localisation of PGs1 at the
centrosome does not involve the putative AKAP binding
domain of the protein.
Three-dimensional organisation of PGs1 on the
To better define the centrosomal area where the endogenous
PGs1 is localised we analysed co-staining images of PGs1 with
polyglutamylated tubulin (Fig. 7A), γ-tubulin (Fig. 7B) and
AKAP-450 (Fig.7C) by deconvolution and 3-D reconstruction.
L83 staining (red) does not overlap with GT335 staining
(green) of the centrioles, but builds an additional layer
surrounding the γ-tubulin and AKAP450 clouds (green)
(Fig. 7A-C). Co-staining with anti-α-tubulin mAb (green,
Fig. 7D) showed that MTs extend within this layer and
revealed, in addition, that smaller dots of PGs1 present in the
cytoplasm are located in the close vicinity of MTs to which
they seem to be associated. These observations culminate with
the suggestion that PGs1 is concentrated around the
centrosome, but not directly anchored to this structure.
Centrosomal localisation of PGs1 is dependent on the
integrity of the MT network
To test if MTs are implicated in the centrosomal localisation
of PGs1, we studied the localisation of PGs1 in cells treated
with drugs affecting the polymerisation state of MTs.
Incubation of 1009 EC cells with 10 µM nocodazole for
45 minutes leads to the depolymerisation of almost all MTs
and is accompanied by the diffusion of PGs1 into the
Journal of Cell Science 116 (20)
Fig. 5. PGs1-GFP localises to the centrosome. (A,B) 1009 cells were
transfected with a PGs1-GFP construct and analysed 24hours after
transfection. Cells were fixed with paraformaldehyde and stained
with L83 (right panel in A) or CTR-453 (right panel in B). Left
panels show PGs1-GFP fluorescence. (C,D), PGs1-GFP fluorescence
of stable transfected 1009 cells, fixed with paraformaldehyde (C) or
unfixed (D). Scale bars: 10µm. (E) Western blotting of 1009 (lane 1)
and stable transfected 1009/PGs1-GFP (lane2) cell extracts with
L83. A single band at 60×103was detected in the latter but not in the
former. Note that the endogenous PGs1 is not detected.
Fig. 6. PGs1 co-localises with PKA. (A,B) Exponentially growing
1009 EC cells were fixed with methanol and double stained with L83
(left panels) and anti-RIIα (right panel in A) or anti-RIIβ (right panel
in B). (C,D) 1009 cells were transfected with a PGs1-GFP construct
and analysed 24 hours after transfection. Cells were fixed with
paraformaldehyde and stained with anti-RIIα (right panel in C) or
anti-RIIβ (right panel in D). Left panels show PGs1-GFP
fluorescence. Scale bars: 10µm.
4187PGs1, a subunit of tubulin polyglutamylase
cytoplasm (Fig. 8A,B). Washing off nocodazole results in a
rapid re-growth of MTs from the centrosomes and a
redistribution of PGs1. 3 minutes after washing, PGs1 is
detected along the length of the newly nucleated microtubules
(Fig. 8C) and begins to concentrate at the centre of the aster.
3 minutes later, PGs1 is almost entirely focused at the centre
of the microtubule network (Fig. 8D). Similar results were
obtained with live 1009/PGs1-GFP cells (Fig. 8F-H).
Delocalisation of PGs1 is also observed when cells are treated
with 5 µM taxol for 45 minutes, but as expected in this case,
PGs1 is found associated with the MT bundles (Fig.8E). From
these observations we conclude that PGs1 is not directly
anchored at the centrosome, but transported towards, and
maintained at, the centrosome via its association with the MT
Localisation of PGs1 is regulated during the cell cycle
In the 1009 EC cell line, endogenous PGs1 is found at the
centrosome in the majority of the cells in interphase but is
hardly detectable in mitotic cells (not shown). Fig.9A,B show
similar results obtained from fixed 1009EC/PGs1-GFP cells.
These observations were confirmed by time-lapse imaging. A
Fig. 7. 3D reconstruction of PGs1 at the centrosome. 1009 EC cells
were fixed with methanol and double stained with L83 (red) and
GT335 (A, green) or GTU-88 (B, green) or CTR-453 (C, green) or
DM1A (D, green). Confocal images were analysed by deconvolution.
Projections of all planes are shown in left panels. 3D reconstructions
are shown in the middle and right panels in two different
orientations. In D (right), the green colour was made semi-
transparent to better visualise the red staining. The arrowheads in D
(left), indicate PGs1 dots associated with MTs.
Fig. 8. Localisation of PGs1 is dependent on the integrity of the MT
network. 1009 cells were incubated with (B) or without (A) 10µM
nocodazole for 45 minutes. After washing off nocodazole, MTs were
allowed to regrow for 3 minutes (C) or 6 minutes (D). (E) 1009 cells
were incubated with 5µM taxol for 45 minutes. Methanol-fixed cells
were double stained with L83 (left panels in A-E) and DM1A (right
panels in A-E). Scale bars: 10µm. (F-H) Confocal green
fluorescence images of living 1009/PGs1-GFP cells before (F) and
after incubation with 10µM nocodazole for 45minutes (G) and after
10minutes of regrowth (H).
typical example, presented in Fig. 9C, shows a cell going
through mitosis. The green label remains focused for 30
minutes and then becomes suddenly faint and diffuse at 40
minutes, when chromosomes appear nicely aligned on the
metaphase plate. These data indicate that de-localisation of
PGs1 occurs at the G2-M transition. As cells exit mitosis
(70 minutes), PGs1-GFP is again detectable and focused,
indicating that after cytokinesis, the protein is rapidly
recruited again to the centrosomes. These observations show
that localisation of PGs1 to the centrosome is cell-cycle
TPG is a multimeric enzyme
In this report, we present evidence that mammalian brain
tubulin polyglutamylase is a complex of at least three major
protein species. These proteins seem to be present in
stoechiometric amounts, indicating that the enzyme complex is
likely composed of two molecules of each subunit. Such a
complex would have a total mass of approximately 324×103)
close to the value of 360×103
sedimentation coefficient (10 S) and Rs (70 Å) in native
conditions (Regnard et al., 1998). We describe here the 32×103
subunit of the complex, called PGs1, in more detail. The two
other proteins co-purifying with PGs1, p50 and p80, were
recently identified as proteins of yet unknown function for
which a detailed analysis is going on in the laboratory.
PGs1 was identified as the product of the gene GTRGEO22
in mouse, initially annotated as encoding for a putative protein
of unknown function. Several lot of data strongly argue for
considering PGs1 as a subunit of TPG. First, antibodies raised
against PGs1 (L83) immunoprecipitate TPG
Conversely, a monoclonal antibody, selected for its capacity
to immunoprecipitate TPG activity, immunoprecipitates PGs1.
Second, PGs1 is enriched during the purification of TPG in
a manner similar to that of TPG activity. Third, PGs1 is
concentrated around known major sites of tubulin
polyglutamylation, namely MTs in neurones, centrosomes,
basal bodies and axonemes. In addition, a recent report shows
that an insertional mutation localised in GTRGEO22 results in
pleiotropic effects, including complete male sterility due to
strong defects in sperm flagella development (Campbell et al.,
2002). Identification of PGs1 as the polypeptide encoded by
this gene strongly argues for a direct role of polyglutamylation
in the morphogenesis of the complex MT assembly of
axonemes. This conclusion is strengthened by previous data
showing that polyglutamylation is actually implicated in
axoneme beating (Gagnon et al., 1996; Million et al., 1999)
and in the stability of centrioles (Bobinnec et al., 1998a).
Recently, TPG from Crithidia was proposed to be a NIMA-
related protein, called CfNek (Westermann and Weber, 2002).
As its most closely related mammalian Nek2 orthologue (Fry
et al., 1995), CfNek exhibits a β-casein kinase activity. The
authors proposed that polyglutamylation could require the
phosphotransferase activity of this protein to generate peptidic
bonds. We found that some α- and β-casein kinase activities
are present in fraction IV, but were lost after TPG pull down
(data not shown), suggesting that brain TPG does not contain
a Nek-related subunit and could follow a different catalytic
mechanism. Mammalian and Crithidia TPGs differ also by
their overall organisation, since the latter has been purified as
a single polypeptide displaying a sedimentation coefficient of
3S (Westermann et al., 1999). Such strong differences are
rather unexpected but could be related to the more complex
functions of polyglutamylation in multi-cellular organisms,
compared to flagellates. Nevertheless, it is interesting to note
determined from the
Journal of Cell Science 116 (20)
Fig. 9. PGs1 disappears from centrosomes at mitosis. (A,B) Fixed
1009/PGs1-GFP cells immunolabelled with CTR-453 (middle
panels). Upper panels show the green fluorescence of PGs1-GFP
and lower panels DAPI staining of DNA. The intensity of labelled
cells in interphase (I), metaphase (M) and anaphase (A) is
compared. (C) Time-lapse video microscopy of 1009/PGs1-GFP
cells. Phase contrast (left panels) and green fluorescence (right
panels) images were taken at 10minutes intervals. Arrows indicate a
cell that goes through mitosis during the time course of the
4189PGs1, a subunit of tubulin polyglutamylase
that CfNek is associated with basal bodies in Crithidia
(Westermann and Weber, 2002) and Nek2 with centrosomes
in higher eukaryotes (Fry et al., 1998), raising the possibility
that the latter kinase could be implicated in the regulation of
some aspects of polyglutamylation.
PGs1 localisation and the mechanism of MT
Overexpression of PGs1 in cultured cells does not lead to any
detectable increase in TPG activity. In particular, no increase
of the polyglutamylation levels of centrosomes and
cytoplasmic microtubules was observed by IF labelling with
GT335. In addition, when cell extracts of 1009/PGs1-GFP
were assayed for in vitro polyglutamylation, no increase over
control non transfected cells was observed. These data suggest
that one or both of the other subunits of the complex are
required for TPG activity and are likely limiting in transfected
cells. Thus PGs1 has no catalytic activity on its own. The
localisation data presented here favour a role of PGs1 in
addressing TPG at particular sites of polyglutamylation.
In neurones, PGs1 is mostly found in the cell body and in
the proximal part of the neuronal processes whereas
polyglutamylated tubulin is highly enriched all along neurites.
This suggests that MTs or tubulin oligomers are
polyglutamylated before or just upon their entry into the
neurites. At the centrosome, PGs1 is found in close proximity,
but not directly in contact, with the strongly polyglutamylated
centriolar MTs. Again, this pattern favours a model of
polyglutamylation of tubulin occurring before its incorporation
into the centriole. In addition, as PGs1 localises at the base of
cilia and in the proximal part of flagella, polyglutamylation
most likely occurs before the transport of tubulin to the tip of
the axonemes where it will be incorporated in the axonemal
structure (Iomini et al., 2001).
However, cytoplasmic MTs nucleating at the centrosome or
present in the vicinity of the base of cilia are not or very poorly
polyglutamylated (Bobinnec et al., 1998b; Million et al., 1999;
Kann et al., 2003). One explanation would be the presence of
a strong deglutamylation activity selectively present in the
cytoplasm, which would counterbalance TPG activity.
Alternatively, we cannot exclude, at present, that the TPG
complex is localised around centrosomes and basal bodies as a
kind of stock pile, from which the core enzyme could dissociate
to selectively interact and polyglutamylate those structures.
PGs1 is addressed at the centrosome in a cell cycle-
In 1009 cells overexpressing PGs1-GFP the majority of the
protein is detected at the centrosome, in a manner
indistinguishable from the endogenous protein. Since the levels
of PGs1-GFP greatly exceed that of the endogenous protein, the
centrosomal localisation of PGs1 is probably independent of the
presence of the other TPG subunits which would be titered out.
However, unlike other centrosomal proteins such as pericentrin
and γ-tubulin (Young et al., 2000), PGs1 is completely
delocalised upon disruption of the MT network, suggesting that
the protein is not anchored at the centrosome. During MT re-
growth, PGs1 rapidly associates with the newly nucleated MTs
and accumulates in the pericentriolar area. This suggests that
PGs1 is transported along MTs by a minus-end-directed motor
activity. 3-D reconstruction of PGs1/tubulin double staining
strongly suggests that, although PGs1 forms a complex matrix
surrounding the centrioles, most elements of this matrix directly
interact with the minus end regions of centrosomal MTs. It is
thus likely that both the transport and the association of PGs1
with centrosomes are directly and exclusively dependent on the
interaction with MTs. The dissociation of PGs1 from
centrosomes observed in mitosis could then be due to the
depolymerisation of the interphasic MT network. Preliminary
experiments performed on nocodazole-arrested mitotic
1009/PGs1-GFP cells, suggest that partial degradation of PGs1
might also occur at the time of mitosis and could prevent re-
association with the mitotic spindle and poles. However, an
additional mechanism avoiding re-association with MTs is
likely to be implicated, since the residual non-degraded PGs1-
GFP present in these cells diffuses in the cytoplasm and does
not localise either on the spindles or at the spindle poles.
It is important to note that PGs1 contains a domain located
in the N-terminal part of the protein (aa 56-85) which shows
homology to a consensus sequence from regulatory subunits of
PKA. This sequence was shown to be responsible for the
dimerisation of PKA and binding of PKA to AKAPs (for
reviews, see Colledge and Scott, 1999; Diviani and Scott,
2001). Our data show that PGs1 follows a particular pathway
for centrosomal localisation, which does not involve the
putative AKAP binding domain. Whether this domain plays
other functions remains to be investigated.
PGs1 and isozymic variants of TPG.
The question of how the different substrates (α-tubulin, β-
tubulin and NAPs) could be specifically polyglutamylated is a
central issue. Brain TPG is not active on NAP substrate (our
unpublished results). It was shown to preferentially
polyglutamulate α-tubulin, but polyglutamylation of the β-
subunit also occurs, albeit at a much lower rate (β:α ratio, 1:3)
(Regnard et al., 1998). In contrast, the major activity present
in HeLa cells strongly polyglutamylates β-tubulin and NAPs,
but not α-tubulin. It is interesting to note that a minor activity
is also present in these cells, with a strong specificity towards
α-tubulin and is indistinguishable from the brain TPG. This
minor type is solubilised only at high salt concentration,
suggesting that it is associated with a rather insoluble structure,
possibly centrosomes (Regnard et al., 1999). We thus propose
the existence of two main polyglutamylase types. TypeI would
be the main type present in proliferative cells and responsible
for the polyglutamylation of β-tubulin and probably NAPs.
Type II would be responsible for the preferential activity
towards α-tubulin observed in neurones, axonemes and
centrioles. The molecular
polyglutamylase is at present not known. It is likely that
specific domains or subunits of the polyglutamylase complex
could be involved in substrate specificity and localisation sites.
The data presented here suggest that PGs1 is specific to type
II polyglutamylase and functions as a localisation subunit.
composition of type I
We gratefully acknowledge the technical contributions of N.
Camcho and C. Pellet. We also thank P. Huitorel for immunolabelling
of spermatozoa, G. Keryer for providing polyclonal and monoclonal
(CTR-453) anti-AKAP450 antibodies, A. Fry for providing Nek2
recombinant protein and anti-Nek2 antibodies, J. Derancourt and E.
Demey for mass spectrometry analysis, J. C. Larcher, P. Steffen and
P. Chaussepied for helpful discussion. This work was supported by
the Association de la Recherche Contre le Cancer (ARC 5859 to B.E.
and ARC 5479 to D.F.) and EMBO Long-term Fellowship (ALTF
387-2001 to C.J.).
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Journal of Cell Science 116 (20)