School of Biological Sciences, University of Manchester, 2.205 Stopford
Building, Oxford Road, Manchester M13 9PT, UK
*
Author for correspondence (e-mail:
k.gull{at}man.ac.uk
)
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SUMMARY |
---|
It has also become increasingly evident over the past year that some (but
intriguingly not all) eukaryotes encode several other tubulin proteins, and to
date five further members of the tubulin superfamily, ,
,
,
and
, have been identified. Although the role of
-tubulin in the nucleation of microtubule assembly is now well
established, far less is known about the functions of
-,
-,
- and
-tubulin. Recent work has expanded our knowledge of the
functions and localisation of these newer members of the tubulin superfamily,
and the emerging data suggesting a restricted evolutionary distribution of
these `new' tubulin proteins, conforms to established knowledge of microtubule
cell biology. On the basis of current evidence, we predict that
-,
-,
- and
-tubulin all have functions associated with the
centriole or basal body of eukaryotic cells and organisms.
Key words: Tubulin, Cytoskeleton, Microtubule, Flagellum, FtsZ, Evolution
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INTRODUCTION |
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`My soul, sit thou a patient looker on;Judge not the play before the play is done:
Her plot hath many changes; everyday
Speaks a new scene; the last act crowns the play.'
Francis Quarles (1592-1644)
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Introduction |
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|
The involvement of microtubules in a wide set of cellular structures and
processes was widely recognised from early studies of their cell biology. This
led to the development of the multi-tubulin hypothesis (Fulton and Simpson,
1976), which acknowledged
tubulin diversity and proposed that distinct microtubule structures within a
cell comprise different forms of tubulin protein. It is now apparent that most
eukaryotic cells can indeed express multiple isotypes of
ß-tubulin
and that this diversity can be further elaborated by a kaleidoscopic array of
post-translational modifications (reviewed by Luduena,
1998
). Although the functional
significance of this diversity has been, and still is, difficult to weave into
a consistent cell biological description, there is increasing evidence that
different
ß-tubulin isotypes and modifications can influence
microtubule structure and function.
It is becoming increasingly evident, however, that eukaryotic cells encode
other tubulin proteins, a further five members of the tubulin superfamily,
,
,
,
and
, having been identified to date
(Chang and Stearns, 2000
;
Dutcher and Trabuco, 1998
;
Oakley, 2000a
; Oakley and
Oakley, 1989
; Ruiz et al.,
2000
; Vaughan et al.,
2000
). Since the
identification of these tubulins comes from work in organisms whose genomes
are not completely sequenced, further members of the tubulin superfamily
probably still await discovery. Here, we review recent work on
- and
ß-tubulin isotypes, both genome encoded and produced by
post-translational modification, but also re-examine the multi-tubulin
hypothesis in the light of the recent extension of the tubulin
superfamily.
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Gene-encoded isotype-specific functions for ![]() |
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It is important to recognise that, by necessity, many of the mammalian cell
and protist studies used proliferating cells. The recognition that divergent
- and ß-tubulin isotypes can participate in different microtubule
structures in such cells was entirely consistent with studies in some
protists. For example, when the amoeba of Physarum differentiate to
form plasmodia, the intermediate cells in transition acquire new microtubule
arrangements quickly, but the tubulin isotype configuration changes only over
a number of cell cycles (Burland et al., 1992). Hence, mixing and co-assembly
of tubulin isotypes would be of particular benefit to cells in such
differentiation transitions.
Molecular studies in Caenorhabditis elegans, however, indicate
that particular tubulin isotypes can influence the supramolecular organisation
of microtubular structures. For instance, the ß-tubulin isotype MEC-7
from C. elegans is expressed primarily in microtubules within the
axons of touch receptor neurons (Hamelin et al.,
1992). Although C.
elegans microtubules normally consist of 11 protofilaments, these axonal
microtubules are structurally distinct and consist of 15 protofilaments. In
mec-7-null mutants, however, microtubules based on 11 protofilaments
are formed, which indicates that the MEC-7 isotype specifically influences the
structural organisation of the axonal microtubule (Savage et al.,
1994
). The
-tubulin
MEC-12 is also required for touch sensitivity and 15-protofilament-microtubule
assembly (Fukushige et al.,
1999
).
Raff and colleagues have provided an elegant dissection of the
ß-tubulin isotype family in Drosophila, revealing
important roles and attributes of specific isotypes particularly in axonemal
morphogenesis (Hoyle and Raff,
1990
; Raff et al.,
2000
; Wilson and Borisy,
1997
). Raff et al. noted that
the presence of the amino acid sequence EGEFXXX (where X is an acidic residue)
close to the C-terminus correlates with the assembly of certain ß-tubulin
isotypes into axonemes (Raff et al.,
1997
). Luduena extended this
analysis to a large number of tubulin isotypes and confirmed the absence of
this sequence in many protists that do not form flagella (yeasts and
filamentous fungi) and its presence in many that do (ciliates, flagellates and
algae; Luduena, 1998
). Some
evidence suggested that this type of sequence is unlikely to operate as a
simple signal sequence for axonemal microtubule assembly itself (Luduena,
1998
). Rather, it may
influence the interaction, and therefore function, of these microtubules with
accessory proteins and, of course, provide target sites for C-terminal
isotype-specific post-translational modifications (see later).
Raff and colleagues have also studied morphogenesis of Drosophila
basal bodies, which contain only the ß1-tubulin isotype, and the sperm
flagellar axoneme, where only the ß2-tubulin isotype is used. They asked
whether ß1-tubulin alone can function in axonemes and found that it
cannot (Raff et al., 2000). In
these males, 9+0 axonemes were initiated at the basal body, and they extended
for only a fraction of the normal length. Males possessing equal amounts of
the isotypes showed equimolar incorporation of both into axonemes. Finally,
increasing the amount of ß1-tubulin produced axonemes that had 10
doublets, and the addition of the extra doublet occurred by a mechanism of
lateral insertion rather than templating at the basal body. There was also
ectopic occurrence of doublets in the cytoplasm, which Raff and colleagues
suggest represents promiscuous initiation, and reflects ß1-tubulin
function in assembly of the triplet microtubules of the basal body. These
studies emphasise the fact that tubulin isotype can matter! Small differences
between two ß-tubulin isotypes influence their ability to form particular
microtubule types. Overall, these studies emphasise the fidelity of axonemal
doublet assembly from the basal body, which is influenced by direct templating
or local concentrations of nucleating molecules. However, the properties of
particular tubulin isotypes (e.g. ß2 but not ß1 tubulin) have an
enormous influence upon assembly of the central pair microtubules and
associated structures.
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Structure of the ![]() |
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This view of the structure of the tubulin dimer has been extended to reveal
the arrangements within the microtubule itself. A high-resolution model of the
microtubule was recently generated by docking of the 3-D crystal structure of
tubulin into a 20 3-D reconstruction of a
microtubule (Amos, 2000
;
Nogales et al., 1999
). This
model predicts the detailed architecture of the microtubule and provides an
insight into the molecular interactions between tubulin molecules (see
Fig. 2). These studies have
revealed that most of the lateral interactions between tubulin protofilaments
occur through the M-loops on one side contacting helix H3 in the adjacent
protofilament (Nogales et al.,
1999
). Structural interactions
between tubulin protofilaments and molecules such as motor proteins and
microtubule-associated proteins can also be predicted from this model (Amos,
2000
).
|
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Post-translational modifications of ![]() |
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Tubulin acetylation
Many isotypes of -tubulin are modified by the addition of an acetyl
group to a lysine residue at position 40. Although the precise function of
acetylated
-tubulin is uncertain, this modification is widespread,
being found on
-tubulin from vertebrates to many protists. Acetylated
-tubulin is associated particularly with stable microtubular
structures, such as axonemes, although this is not an invariant rule
for instance, in trypanosomes the ephemeral microtubules of the mitotic
spindle are acetylated. Acetylation is a post-assembly phenomenon (Sasse and
Gull, 1988
), and the general
correlation with stable microtubules is really a reflection of the relative
length of time that the individual microtubule subunits are presented as a
substrate for tubulin acetyltransferase (Maruta et al.,
1986
). Molecular engineering
of protists has shown that organisms that normally express lots of acetylated
-tubulin can use non-acetylated tubulin (Rosenbaum,
2000
). Engineering of
Tetrahymena to express
-tubulin that cannot be acetylated at
Lys40 produced mutant cells devoid of acetylated tubulin but expressing no
novel phenotype (Xia et al.,
2000
). Tetrahymena is
proving to be an excellent organism for such studies, because it constructs
its microtubules from single
- and ß-tubulin nascent isotypes.
These studies suggest that acetylated
-tubulin is not important under
the conditions tested or alternatively that other events/proteins can supplant
the normal function. However, Stanchi et al. recently described a novel,
tissue-specific isoform of
-tubulin in human and mouse (Stanchi et al.,
2000
) that is highly divergent
around the acetylation motif. This isotype does not possess the Lys40 residue
necessary for acetylation, and the amino acid sequence from position 32 to 45
is also highly divergent from all other human/mouse isotypes. The intriguing
possibility that this isotype has evolved in a cellular environment in which
the acetylation motif is released from normal constraints needs to be
addressed.
Tubulin C-terminal modifications
Interestingly, many tubulin modifications occur in the exposed acidic
C-terminal domain of ß-tubulin, the region that also provides the
major source of genetically encoded isotypic
- and ß-tubulin
variation. These modifications include the tyrosination/detyrosination of
-tubulin and the polyglutamylation and polyglycylation of both
-
and ß-tubulin. Unfortunately, these regions are not resolved in the
crystal structure of tubulin, but their positions in the model strongly
suggests that they are exposed on the outer surface of the microtubule
(Nogales, 2000
).
The tubulin tyrosination cycle involves the enzymatic removal of the
C-terminal tyrosine residue present on some -tubulin isotypes by a
specific carboxy-peptidase, and its subsequent restoration by a
tubulin-tyrosine ligase (reviewed by Idriss,
2000
). Although the functional
relevance of this modification is not always clear, highly stable microtubules
such as those of the axoneme are detyrosinated, and this appears to reflect
the length of time the individual
-tubulin substrate molecule has spent
in a microtubule. The trypanosome cytoskeletal microtubules provide an
opportunity for visualisation of this process along individual microtubules
(Sherwin and Gull, 1989
), and
there are extensive descriptions of the distribution of detyrosinated
-tubulin in microtubule arrays. The tubulin-tyrosine ligase enzyme, its
gene and related genes have been characterised (Ersfeld et al.,
1993
; Trichet et al.,
2000
). Although we still do
not fully understand the functional implications of the tubulin tyrosination
cycle, recent evidence indicates that detyrosination of tubulin can regulate
interaction of microtubules with vimentin intermediate filaments by a
kinesin-dependent mechanism (Kreitzer et al.,
1999
).
Removal of the penultimate glutamate residue from the -tubulin
polypeptide produces
2-tubulin, a derivative that is unable to act as a
substrate for tubulin-tyrosine ligase, and this truncated protein is therefore
removed from the tyrosination cycle.
2-tubulin is particularly
prevalent on microtubular structures such as the axonemes of flagella and
cilia and also in mammalian brain cell microtubules.
The tubulin modifications polyglutamylation and polyglycylation involve the
attachment of oligoglutamyl and oligoglycyl side chains of variable length to
specific glutamate residues located near the C-terminus of both - and
ß-tubulin. These side chains can be of considerable length for instance,
axonemal tubulin of Paramecium is modified by up to 34 glycyl
residues (Bre et al., 1998
),
and the microtubules of Trypanosoma brucei contain 15 glutamyl
residues per
-tubulin subunit (Schneider et al.,
1997
). Both
- and
ß-tubulin can be simultaneously modified by both polyglycylation and
polyglutamylation (Mary et al.,
1996
), as well as being
glutamylated at multiple sites (Schneider et al.,
1998
).
Polyglutamylation and polyglycylation are again particularly associated
with stable microtubule structures such as the axonemes of cilia and flagella.
Antibodies specific for these modifications inhibit the beating of flagella
and cilia, implicating polyglutamylation and polyglycylation in regulation of
axonemal function (Gagnon et al.,
1996).
The axonemes of virtually all eukaryotic cilia and flagella are remarkably
similar in their organisation, consisting of nine outer doublet microtubules
(designated the A and B tubules) surrounding a central pair of singlet
microtubules. Studies on the axonemes of sea urchin sperm have also shown that
that A and B tubules have differing degrees of tubulin modification (Multigner
et al., 1996). The extent of
tubulin polyglycylation and polyglutamylation also varies in a gradient along
the flagellum, so that a variety of structurally distinct regions appear to
exist within the flagellum/cilium axoneme in different systems (Pechart et
al., 1999
). Recently,
differences have been noted between the modifications present in centriolar
microtubules and those of the axoneme. Centriolar microtubules appear to be
polyglutamylated but not polyglycylated (Million et al.,
1999
). Polyglutamylation
appears to be critical for the stability of centriole microtubules, since
microinjection of monoclonal antibodies specific for polyglutamylated tubulin
isotypes, results in the transient disappearance of centrioles in mammalian
cells (Bobbinec et al.,
1998
).
Polyglutamylation also represents the major post-translational modification
of axonal tubulin in neuronal cells, where it appears to regulate the
differential interaction between microtubules and microtubule-associated
proteins (MAPs). For instance, MAPs such as Tau and kinesin exhibit optimal
binding to tubulin modified by 3 glutamyl residues, binding affinity
decreasing with increased polyglutamyl chain length (Boucher et al.,
1994
; Larcher et al.,
1996
). In contrast, increasing
polyglutamyl chain length does not appear to affect the binding affinity of
MAP1A significantly (Bonnet et al.,
2001
). Bonnet et al. suggest
that the differential binding of MAPs to polyglutamylated tubulin could
facilitate their selective recruitment to distinct microtubule populations and
thereby modulate the functional properties of microtubules.
The importance of the polyglycylation modification has also been dissected
by molecular genetic approaches in Tetrahymena (Xia et al.,
2000), in which, as we have
discussed above in the context of tubulin acetylation, one can engineer
modifications to post-translational modification sites in both
-and
ß-tubulin. Modifying the multiple polyglycylation sites in
-tubulin, such that the modified tubulin isotype was unable to form in
the mutant cell, produced no observable phenotype. In contrast, ß-tubulin
polyglycylation was essential. However, reducing but not eliminating
polyglycylation of ß-tubulin, by modifying a proportion of the sites,
resulted in slow growth, reduced motility and defects in cytokinesis.
Interestingly, a double mutant that has a fully non-glycylated ß-tubulin
and an
-tubulin that has a wild-type C-terminus from ß-tubulin is
viable. This
-ß chimeric tubulin becomes hyperglycylated, which
suggests that it is the level of polyglycylation modification rather than the
specific isotype that is important.
Although this series of elegant experiments goes a long way to providing
explanations for tubulin modifications (Rosenbaum,
2000), we probably still have
some way to go to fit the explanation into a global context. The evidence
regarding essential functions of polyglycylation revealed in
Tetrahymena can be considered in evolutionary terms. Polyglycylation
does not appear to be a universal modification of tubulin. The general
observation is that most of the modifications appear to have developed early
in the evolution of eukaryotic cells and are present in many protists. A
comparison of representatives of three ancient groups the trichomonads
(Tritrichomonas mobilensis), the trypanosomatids (T. brucei)
and the diplomonads (Giardia lamblia; Schneider et al.,
1998
; Schneider et al.,
1997
; Weber et al.,
1997
) is informative
in this respect. Tubulin acetylation and tubulin polyglutamylation are present
in all three, but tyrosination has been detected only in trypanosomes and may
therefore have appeared after the trichomonads diverged from the rest of the
eukaryotes. Conversely, polyglycylation has been detected in the diplomonads
and not in the other two groups, which suggests it is an ancient modification
that was subsequently lost in trichomonads and trypanosomatids. So, although
polyglycylation appears to be an essential function in the ciliate
Tetrahymena, the ancient flagellate T. brucei appears to be
able to operate without it.
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The extended tubulin superfamily |
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|
-tubulin
-tubulin was first identified in the filamentous fungus
Aspergillus nidulans as a result of a genetic screen designed to
identify proteins that interact with ß-tubulin (Oakley and Oakley,
1989
). mipA encodes a
protein that was clearly a new member of the tubulin family and was termed
-tubulin. It is approximately 30% identical to
- and
ß-tubulin, has subsequently been described in a wide variety of
eukaryotic organisms and is likely to be present in all eukaryotes.
-tubulin is located in centrosomes and other MTOCs, such as the spindle
pole body (SPB) of fungi, where it plays an essential role in initiation of
microtubule assembly. Eukaryotic cells, however, contain a large amount of
soluble
-tubulin. Some is associated with the TCP-1 chaperonin complex,
but much is associated with evolutionary conserved protein complexes
(
-TuRC and
-TuSC) that play a central role in nucleation of
assembly at the minus end of microtubules (Oakley,
2000a
).
-tubulin
The gene encoding -tubulin was also first identified by genetic
means, as a mutation resulting in defective basal body function in the green
alga Chlamydomonas (Dutcher and Trabuco,
1998
). The mutation results in
elevated frequencies of formation of uniflagellate cells, and their flagellar
basal bodies possess doublet rather than the typical triplet arrangement of
microtubules, owing to the specific loss of the C-tubule. Recently Garreau de
Loubresse et al. have shown that inactivation of
-tubulin in
Paramecium results not only in the loss of the C-tubule but also in
the loss of the B- and A-tubules at one or more triplet sites within the basal
body (Garreau de Loubresse et al.,
2001
).
Database searches have also now identified -tubulin in humans, mice,
rats and trypanosomes (Chang and Stearns,
2000
; Vaughan et al.,
2000
), and we have observed
that it is also present in other protists, such as the malarial parasite
Plasmodium. Immunofluorescence detection of
-tubulin in
Chlamydomonas suggests that it is localised to the basal body area
(Dutcher, 2001
). Chang and
Stearns immunolocalised human
-tubulin to a centrosomal region between
centrioles and also to an intercentriolar location between duplicated
centrosomes (Chang and Stearns,
2000
). However
immunolocalisation of mouse
-tubulin in somatic cells (Smrzka et al.,
2000
) suggested that this
protein has a cytoplasmic and nuclear location, being only particularly
enriched at spindle poles during mitosis. In contrast, in mouse sperm cells,
-tubulin is associated with the perinuclear ring of the manchette,
centriolar vaults and the flagellum.
Understanding -tubulin function in flagellated and nonflagellated
cells will require ultrastructural localisation of the protein. This fine
level of structural information will also probably be required for the other
members of the extended tubulin superfamily that seem to have restricted
distributions. Obviously, as more tubulins come to light, it makes the
production of specific antibodies all the more necessary and all the more
difficult!
-tubulin
-tubulin was discovered independently in mammalian cells by genomic
approaches (Chang and Stearns,
2000
) and in trypanosomes by a
combination of genomic and cloning survey approaches (Vaughan et al.,
2000
). Database searching
shows that this new tubulin is present in many but not all eukaryotes (see
below). Immunolocalisation of
-tubulin and GFP-tagged
-tubulin in
mammalian cells shows it to be located to the centriolar area, and this
localisation shows cell-cycle-dependent modulation. During the early phase of
the cell cycle,
-tubulin associates predominantly with the old
centrosome and only later in the cell cycle becomes associated with both the
old and new centrosome (sometime after centrosome segregation). Mammalian cell
centrosomes clearly exhibit cell-cycle-dependent protein patterns that reflect
the structurally complex nature of replication and maturation of centrioles
within the centrosome (Lange et al.,
2000
; Lange and Gull,
1995
; Lange and Gull,
1996
; Tassin and Bornens,
1999
). Immunolocalisation at
higher resolution will be required if we are to unravel how the intricacies of
-tubulin positioning at the centrosome relate to the replication and
maturation of the centrioles at the centrosome core. Although the role of
-tubulin is at present unresolved, Chang and Stearns have observed that
centrosomes can nucleate microtubule assembly irrespective of
-tubulin
content, which indicates that
-tubulin is not involved in microtubule
nucleation (Chang and Stearns,
2000
).
-tubulin
-tubulin was discovered in trypanosomes, and the only full-length
sequences available so far are from T. brucei and Leishmania
major (Vaughan et al.,
2000
). However, there is
evidence (see below) from database searches that this branch of the tubulin
superfamily also exists in other organisms (Dutcher,
2001
). Immunofluorescence and
immunoelectron microscopy has revealed that
-tubulin localises to the
basal body region in trypanosomes and at the centriolar region in some animal
cells (S.V., A. Baines, P.G.M. and K.G., unpublished).
-tubulin
- tubulin was discovered by genetic means in Paramecium and
is encoded by the SM19 gene (Ruiz et al.,
2000
). The sm 19-1
mutation in Paramecium prodces a cell phenotype in which basal body
duplication is inhibited, the oral apparatus is reduced and
-tubulin
becomes mislocalised. Although we have no precise localisation of
-tubulin so far, the mislocalisation of
-tubulin in sm
19-1 mutants may indicate that
-tubulin tethers
-tubulin (or
-tubulin complexes) to the basal body. Ultrastructural studies of
sm 19-1 mutants reveal a rare, but perhaps important, defect in some
basal bodies that lack microtubules from their triplets (3% of cross sections
of basal bodies). Interestingly, the Paramecium
-tubulin
sequence shows an extreme C-terminus that resembles that of
-tubulin
(it contains a C-terminal EY dipeptide; see below).
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FtsZ |
---|
![]() |
Sequence gazing |
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|
The monoclonal antibody YL1/2 is widely used to detect the presence of
tyrosinated -tubulin in microtubule structures, because it recognises
an epitope minimally dependent on a C-terminally located tyrosine (or
phenylalanine) residue preceded by aspartate or glutamate residue (Wehland et
al., 1984
). The existence of
such a putative C-terminal epitope (-EY) in Paramecium
-tubulin
is intriguing, particularly given that the
-tubulins of
Paramecium lack the C-terminal epitope necessary for recognition by
YL1/2. It has often been observed that this antibody stains basal bodies or
centrioles in some organisms. This monoclonal might be detecting
-tubulin
alone and/or tyrosinated
-tubulin at these sites in such organisms. As
stated earlier, however, the discovery of these new tubulins indicates that,
until the reactivities of antibody probes towards all tubulins are
established, care must be taken in interpretation of such studies.
Structural comparisons
We have manually aligned the six full-length members of the tubulin
superfamily identified in the African trypanosome, T. brucei
(Fig. 3). Our alignment
indicates that there are a number of insertions and deletions in the -,
- and
-tubulin sequences when compared with
ß-tubulin. Insertions and deletions that are >5 residues in
length are highlighted in Fig.
3 and shown schematically in
Fig. 4. The secondary structure
of pig
-tubulin has also been added to indicate where these insertions
and deletions might be located. Although this alignment takes into account
this structure, so that insertions and deletions are restricted to loop
regions wherever possible, some deletions appear to result in a disruption of
the predicted secondary structure. This is most clearly exemplified in the
-tubulin sequence, in which a predicted deletion towards the
C-terminus could result in the loss of H10. In the
ß-tubulin
sequence, H10 is predicted to be one of the regions that makes lateral
contacts between protofilaments and longitudinal dimer contacts at the minus
end surface of tubulin (Inclan and Nogales,
2001
). However, given the
current dearth of knowledge of
-tubulin function, the significance of
this potential deletion is unclear.
|
Although it is evident from the alignment shown in
Fig. 3 that the -,
- and
-tubulin sequences contain a significant number of
insertions and deletions, our analysis suggests that key regions involved in
nucleotide binding (loops T1-T7) are conserved in all of the tubulin
sequences. However, from this sequence gazing, we predict that while
-
and
-tubulin could hydrolyse GTP,
-tubulin does not. We base
this prediction on the observation that, from our alignment, Glu254 in
-tubulin is conserved in the T. brucei
- and
-tubulin. This residue, which is conserved in all
-tubulins, is
essential for GTP-hydrolytic activity. These results are in accord with the
analysis of the human
- and
-tubulin sequence by Inclan and
Nogales, who also recently proposed that
- and
-tubulin could
probably hydrolyse GTP (Inclan and Nogales,
2001
). We would point out that
Inclan and Nogales suggested that the T. brucei
-tubulin was
divergent at this key residue (since it possesses an alanine instead of a
glutamate residue); however, our alignment indicates that this glutamate
residue is also conserved in T. brucei.
We have also noted that the -,
- and
-tubulins show
more variation in sequence than
ß-tubulin. When the first
protistan ß-tubulins were sequenced in the 1980s, much discussion
initially concentrated on the observation that ß-tubulins participating
in the construction of the complex axoneme/basal bodies of flagella and cilia
appeared to be less divergent than those that do not participate (see, for
instance, Singhofer-Wowra et al.,
1986
). This theme can be
considered again in terms of these new tubulins, in the rather different
context of participation in the construction of the main wall of a microtubule
or particular MTOC. Naturally, there are other explanations possible and
caveats even to this one. However, the finding that at least six diverse
members of the tubulin superfamily are present in one trypanosome cell
suggests cell biological distinctions in their functions. Whatever the precise
mechanism of microtubule nucleation (Erickson,
2000
),
-tubulin is
acknowledged to be a universal component of MTOCs (Oakley,
2000b
). A distinct possibility
is that these new tubulins are components of MTOCs or particular subsets of
microtubules.
Using the structure of ß-tubulin as a model, Inclan and Nogales
analysed potential interactions of
-,
- and
-tubulin with
the
ß-tubulin microtubule (Inclan and Nogales,
2001
). Sequence comparisons of
-, ß-,
-,
- and
-tubulin showed clear
conservation of sequence in certain regions, leading to the speculation that
functionally important contacts were being maintained. From this
sequence/structure analysis, Inclan and Nogales suggested that
-tubulin
interacts longitudinally with
-tubulin at the minus end of the
microtubule whereas
-tubulin binds to the plus end of ß-tubulin and
acts as a microtubule-plus-end-capping protein. Similar analysis of the
- and
-tubulin sequences has not yet been undertaken. These in
silico studies are extremely useful in predicting potential interactions of
these new tubulins with the
ß-tubulin microtubule, although
possible interactions with
-tubulin or other protein components of the
centrosome or basal body also need to be addressed.
We have noted previously the unusual distribution of the new tubulins in
eukaryotic genomes and a general correlation with triplet basal bodies or
axonemes (Vaughan et al.,
2000). Certainly, evidence now
indicates that
-,
-,
- and
-tubulin all localise to,
or affect functions at, the centriole or basal body area of cells (Chang and
Stearns, 2000
; Dutcher,
2001
; Ruiz et al.,
2000
). We discuss below how
the evolutionary distribution of the tubulin superfamily members fits with
known microtubule cell biology. In the context of tubulin sequence gazing, we
note that some of those organisms that do not possess
-,
-,
- and
-tubulins, such as yeast and C. elegans, are
organisms whose
-tubulins are rather divergent (Burns,
1995
; Keeling and Logsdon,
1996
; Spang et al.,
1996
). One constraint on the
-tubulin sequence might be the need to interact with
-,
-,
- and
-tubulin in particular MTOCs. Organisms such as yeast would
be released from such constraints, and their
-tubulin sequences could
have diverged as a consequence.
![]() |
An evolutionary and functional survey of the extended tubulin superfamily |
---|
As we discussed above, there is a reasonable correlation between the
occurrence of the new tubulins (,
,
and
) and the
appearance of a motile axoneme and triplet microtubule basal body or
centriole. The yeasts and plants are well known for their lack of such
structures, whereas C. elegans has amoeboid sperm, and the sensory
cilia of nematodes lack normal triplet microtubule basal bodies or centrioles
(Albertson and Thomson, 1993
;
Perkins et al., 1986
; Wolf et
al., 1968
). Although
centrioles that have nine triplets have been reported in Drosophila
(Gonzalez et al., 1998
;
Mahowald and Strassheim,
1970
), variations in which the
centrioles of Drosophila embryos have only doublet microtubules are
known to occur. Callaini has debated these structures in insects and points
out the possibility that `despite the vast literature on insect sperm
structure, it is unclear whether these cells have a true centriole' (Callaini
et al., 1999
). Microtubule
doublets rather than triplets are commonly found in spermatocytes, and there
have been erroneous interpretations of centriole structures in insects, since
accessory microtubules around the axoneme often extend close to and even
beyond the doublets. Hence, even if Drosophila has triplet
microtubules in its centriole, they might not share all the attributes of more
usual triplet microtubule centrioles or basal bodies.
An important question to be addressed, therefore, is why the centrosomes of
Drosophila can dispense with these new tubulins. - and
-tubulin cannot be the result of recent divergence in the tubulin
superfamily, because these tubulins are present in the more evolutionarily
ancient protozoan T. brucei. Greater knowledge of the molecular
functions of
- and
-tubulin, as well as more detailed studies on
the Drosophila centrosome, is undoubtedly required if we are to
understand why these proteins are absent from the Drosophila
genome.
The new tubulins are certainly not restricted to the protists, because full
length - and
-tubulin have been recognised in mammals (Chang and
Stearns, 2000
; Smrzka et al.,
2000
). It is perhaps slightly
dangerous to identify certain tubulin sequences in genomes represented only by
GSS or EST markers. However, both
- and
-tubulin appear to be
represented in Xenopus (Dutcher,
2001
), and there are good
markers for
-tubulin in these and other metazoan databases. Such EST
databases provide us with intriguing glimpses of the evolutionary landscape,
but full characterisation awaits the appearance of complete sequences and
their analysis by a variety of bioinformatics approaches (Attwood et al.,
2000
; Vaughan et al.,
2000
).
Does this glimpse of the extended tubulin superfamily help our conjectures
on function? We have rehearsed above our views on the correlation between
possession of 9+0 triplet basal bodies or centrioles and these new tubulins.
Thus, possession of these new tubulins (,
,
,
) may
indeed relate to construction of complex centrioles or basal bodies. However,
it is important to note other attributes of such organelles apart from their
intrinsic organisation. Defined patterns of centriole/basal body duplication
and maturation are exemplified in protists such as trypanosomes,
Paramecium and Chlamydomonas but are also seen in the
centrioles of mammalian cells. Moreover, some eukaryotic cells are able to
divide while expressing flagella/cilia, whereas others only exhibit
cilia/flagella in differentiated, non-dividing cells such as sperm or ciliated
epithelia. An important function of the new tubulins might be to endow the
basal bodies and centrioles of dividing organisms with properties that
facilitate their dynamic duplication, maturation and inheritance in certain
cells.
Finally, many centrioles, basal bodies and axonemes exhibit very particular
appendages and a circumferential polarity or anisotropy. Although there is
little direct proof of biochemical non-equivalence of triplets (Beisson and
Jerka-Dziadosz, 1999), there
are very good structural examples that suggest non-equivalence of triplets in
centrioles or basal bodies and doublets in the axoneme. Many of these
structural anisotropies of the basal bodies have consequences for the precise
positioning of accessory microtubule arrays emanating from these basal body
MTOCs. We have long recognised the structural complexity of the basal body
region (often most clearly seen in protists) as an integrator of cytoplasmic
form through its nucleation of microtubule arrays. This area of eukaryotic
cells may now be revealing its biochemical complexity.
![]() |
The tubulin superfamily saga |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
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