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INTRODUCTION |
Microtubule filaments are essential components of the eukaryotic
cytoskeleton. For example, they distribute organelles and mRNAs to
various subcellular locales, form the mitotic spindle, and provide the
structure for cilia, flagella, and axons. The microtubule filament
consists of a hollow cylinder composed of 13 protofilaments. These
protofilaments are in turn composed of many
- and
-heterodimers
arranged in a head to tail fashion. In most cells, the number,
orientation, and nucleation of microtubules are controlled by a
discrete structure, the microtubule organizing center
(MTOC)1 (1). The nongrowing
(minus) end of the filaments remains attached to the MTOC, whereas the
growing (plus) end extends away from the MTOC (2).
De novo microtubule synthesis (that is, synthesis from
soluble 7 S
- and
-heterodimers) occurs at the MTOC. In the past few years, much information has been gathered as to how this process occurs. In cycling animal cells, the main MTOC is the centrosome, a
nucleus-associated organelle that consists of centrioles and pericentriolar material. Among the many centrosomal proteins is a
member of the tubulin family,
-tubulin (3, 4). This protein is
evolutionarily conserved, occurring in animals, insects, plants, and
fungi (3, 5-13).
-Tubulin is not an obligate centrosomal component
(14, 15); yet recruitment of
-tubulin to the centrosome is essential
for microtubule nucleation in vivo and in vitro
(5, 8, 9, 11, 14, 16). That the sequence and subcellular localization
of
-tubulin are evolutionarily conserved and that
-tubulin is
essential for microtubule nucleation suggest a universal mechanism in
which
-tubulin plays an essential, universal role at the MTOC,
possibly by nucleating microtubule growth by directly interacting with
tubulin heterodimers.
In cells, cytosolic (that is, soluble, noncentrosomal)
-tubulin is
always found as a large 25 S, ring-like complex (termed a gammasome or
TuRC) that nucleates microtubule assembly when recruited to the
centrosome (13, 17, 18). That
-tubulin is always found in the
gammasome has led to the inference that
-tubulin must interact with
other gammasomal proteins to exert its function. Although some proteins
that might aid
-tubulin in its role have been identified, no direct
evidence has been provided proving their function. For example, the
gammasome includes
- and
-tubulin (13). Also, an additional
protein that may interact with
-tubulin at the centrosome has been
recently identified (19). However, exactly how
-tubulin interacts
with other proteins to become incorporated into the centrosome is
unknown.
In cells, 7 S tubulin heterodimers are added to the growing filament at
the centrosome. That growing microtubules usually have long, curved
sheet-like extensions at their plus end has led to the belief that
soluble, 7 S
- and
-heterodimers are added to the free end of
two-dimensional sheets whose lateral ends eventually circularize to
form a cylinder (20) such that
-tubulin subunits are adjacent to
-tubulin subunits of another protofilament (21-24). However,
exactly how the
-
-heterodimer interacts with other proteins to
become stably incorporated into the growing filament is unknown.
Tubulin proteins show sequence similarity, yet are targetted to
different subcellular localizations and possess different functions.
The basis for these differences must lie at the level of amino acid
sequence, which must specify the protein-protein interactions necessary
for executing tubulin function. Throughout evolution, each tubulin type
shares approximately 65-70% sequence conservation (25, 26). On the
other hand
-,
-, and
-tubulin share only about 30% sequence
identity (25). Between tubulins, there is considerably more sequence
similarity at the amino termini than at the carboxyl termini. This
observation has led to the hypothesis that whereas the amino terminus
of tubulins contains information that pertains to a shared function
among tubulins, the carboxyl terminus contains information that is
specific to the function of a particular tubulin type (25). Clearly,
tubulins play an essential role in microtubule polymerization and
maintenance. To gain new insights into tubulin function, we decided to
determine the roles of the amino and carboxyl termini of
- and
-tubulin in their subcellular localizations and microtubule filament
formation. To address this issue, we expressed epitope-tagged
- and
-tubulin mutants in permanently transfected Chinese hamster ovary K1
(CHO-K1) epithelial cell lines. Using immunofluorescence microscopy,
gel filtration chromatography, and sucrose gradient centrifugation, we
have assayed the cell biological and biochemical properties of these
mutants and have identified regions necessary for microtubule polymerization.
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MATERIALS AND METHODS |
DNA Subcloning--
DNA constructs were made using the
polymerase chain reaction with vent polymerase (New England Biolabs,
Beverly, MA) and purchased DNA primers (Life Technologies, Inc.) as
described (27). Primers were designed such that that fragment could be
cut with HindIII and SalI (New England Biolabs)
and be subcloned into the HindIII and SalI sites
of the vector pTS335 (a derivative of pCDNA I Neo; Invitrogen, San
Diego, CA).2 In the case of
mutants in which the amino-terminal thirds were switched, primers were
generated that contained a silent mutation that introduced a
BstEII site. Thus, hybrids were generated by a three-piece
HindIII/BstEII/SalI ligation.
Fragments were subcloned upstream in-frame to the myc
epitope tag and downstream of the strong viral CMV promoter, enabling
expression of a carboxyl-terminal myc-tagged protein.
Correct sequence of the constructs was verified using Sequenase (U. S.
Biochemical Corp.) as described by the manufacturer.
Cell Culture and Transfection--
CHO-K1 cells (American Type
Culture Collection, Rockville, MD) were cultured in F-12 medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.).
Plasmids were prepared as described by the manufacturer (Qiagen, Santa
Clarita, CA). Cells were transfected with Lipofectin as described by
the manufacturer (Life Technologies, Inc.), and stable transfectants
were selected for and maintained in 800 µg/ml G418 (Life
Technologies, Inc.).
Immunofluorescence Microscopy--
Cells were grown on glass
coverslips (VWR, San Francisco, CA). Cells on coverslips were fixed in
20 °C methanol for 20 min, washed twice in PBS, and blocked at
room temperature for 30 min in PBS that contained 3% bovine serum
albumin and 0.1% Triton X-100. Primary antibodies were then added to
fresh solution and incubated for 30 min. Polyclonal antibodies used (at
1:300 dilution) were: anti-
-tubulin (XGC1-4, raised against a
region in the carboxyl terminus of Xenopus
-tubulin; Ref.
3); anti-
-tubulin (YL1/2, raised against the entire rat
-tubulin
protein; Sera Labs); and anti-
-tubulin (Boehringer Mannheim). The
monoclonal anti-myc antibody was used at 1:300 dilution
(9E10). After washing coverslips three times in PBS, coverslips were
incubated in a fresh solution containing a 1:300 dilution of secondary
Texas Red or fluorescein isothiocyanate-conjugated secondary antibodies
(Jackson Laboratories, West Grove, PA) for 30 min each before washing
and mounting. Nuclei were stained in
4,6-diamidino-2-phenylindone. Cells were examined using an
immunofluorescence microscope (model Axioskop, Carl Zeiss, Inc.,
Thornwood, NY).
Polyacrylamide Gel Electrophoresis and Immunoblot
Analysis--
Cell extracts were prepared by washing cells twice in
PBS and by solubilizing cell pellets in 8 M urea.
SDS-polyacrylamide gel electrophoresis was performed as described using
8.5% polyacrylamide gels (27). Gels were blotted onto nitrocellulose
and blocked overnight at 4 °C in 5% nonfat dry milk, 1 × PBS,
0.1% Tween 20. Blots were incubated for 1 h at room temperature
with each antibody, diluted in 1% milk, 1 × PBS, 0.1% Tween 20. Proteins were detected using a chemiluminescent kit as described by the
manufacturer (Renaissance, NEN Life Science Products). Antibodies used
at 1:1000 dilution were: anti-
-tubulin (XGC1-4; Ref. 14);
anti-
-tubulin (YL1/2); anti-
-tubulin, and anti-myc.
Secondary antibodies (used at a 1:10,000 dilution) were conjugated with
horseradish peroxidase (Jackson).
Gel Filtration and Sucrose Gradient Analysis--
Cell extracts
for gel filtration were prepared by hypotonic lysis. Briefly, cell
pellets were washed twice in PBS and incubated in 10 mM
NaCl, 50 mM Tris-HCl, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, pH 7.5, at 4 °C,
followed by Dounce homogenization. Extracts were subsequently adjusted
to 1 M NaCl. Gel filtration was performed at 4 °C using
a Pharmacia fast performance liquid chromatograph with a Superdex 200 column (Pharmacia Biotech Inc.), with a flow rate of 0.25 ml/min. To
verify molecular masses corresponding to peaks coming off the column,
fast performance liquid chromatography molecular mass standards
(Pharmacia) were run with each experiment. Columns were run and loaded
in 1 M NaCl, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5. Fractions (0.5 ml) were collected and concentrated using
Microcon 10 concentrators (Amicon, Beverly, MA). SDS sample buffer was
added to the samples, which were analyzed using SDS/polyacrylamide gel
electrophoresis.
Extracts for sucrose gradients were performed by lysing cells in 150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA,
2 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, pH
7.5. Sucrose gradient (10-40%) analysis was performed in buffer with
150 µg of extract as described (14). Fractions were collected (either
100 or 150 µl) and processed by SDS/polyacrylamide gel
electrophoresis. Molecular mass standards (Pharmacia) were run in
parallel to each gradient.
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RESULTS |
myc-tagged Human
-Tubulin Is Correctly Localized to the
Centrosome--
Previously, we had demonstrated the importance of
-tubulin in initiating microtubule assembly at centrosomes (14). To
assess how
-tubulin is selected to be localized to the centrosome,
we subcloned full-length human
-tubulin upstream in-frame to the myc epitope tag and downstream of the strong viral CMV
promoter (Ref. 28; Fig. 1). Introduction
of the myc tag at the carboxyl terminus ensured that the
introduced, mutant protein could be detected by immunofluorescence and
Western blotting by using an anti-myc antibody. Cell
extracts from cell lines were subjected to Western blotting to verify
expression of the introduced protein and to verify that the protein
could be detected before cells were processed for immunofluorescence.
Thus, upon transfection of constructs into mammalian cells, high levels
of transgenic protein expression was achieved, and the subcellular
localization of the introduced protein could be readily detected by
immunofluorescent staining with an anti-myc antibody.

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Fig. 1.
Schematic diagram of basic plasmid used in
our transfection experiments. Versions of human - and mouse
-tubulin genes, both mutant and wild-type, were generated by
polymerase chain reaction as described under "Materials and
Methods" and were subcloned into the vector HindIII and
SalI restriction endonucleases. Genes were subcloned into
pTS335 (see "Materials and Methods") downstream of the strong viral
CMV promoter and upstream in-frame to the myc epitope tag,
enabling detection of the transfected protein by Western blot and
immunocytochemical analyses. Line with arrow, CMV promoter;
H3, HindIII restriction endonuclease site;
Sal, SalI restriction endonuclease site;
black box, myc tag; gray box, G418
resistance gene; striped box, kanamycin resistance gene; open box, subcloned gene of interest.
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Because plasmids used in our study contained a gene conferring
resistance to neomycin, stably transfected cell lines could be isolated
by growing cells in the drug G418. Possessing permanent cell lines
expressing
-tubulin was a necessary prerequisite for studying
transfected mutant protein because transiently transfected
-tubulin
was dispersed throughout the cytoplasm. CHO-K1 epithelial cells were
transfected with a construct encoding full-length myc-tagged
-tubulin. Permanent cell lines were generated. From these, cell extracts were prepared and subjected to polyacrylamide gel
electrophoresis. Gels were blotted onto nitrocellulose and probed with
anti-myc and anti-
-tubulin antibodies. Anti-
-tubulin
antibody detected the endogenous
-tubulin in both untransfected and
transfected cell extracts (Fig. 2,
lanes 1 and 2). In extracts prepared from stably
transfected cells, a protein of slower mobility corresponding to
myc-tagged
-tubulin was detected with both
anti-
-tubulin (Fig. 2, lane 2) and anti-myc
antibody (Fig. 2, lane 2), verifying expression of the
transgenic protein. The
-tubulin-myc protein was
correctly localized to the centrosome of these cells as detected by
immunofluorescence staining with an anti-myc antibody; the pattern of staining was indistinguishable from anti-
-tubulin staining of untransfected cells (Fig. 3,
A-D). Because epitope-tagged
-tubulin was correctly
localized to the centrosome of stably transfected CHO-K1 cells, we
decided to delineate domains of human
-tubulin essential for its
subcellular localization by introducing deletions into the amino and
carboxyl termini of
-tubulin and assaying their behavior in stably
transfected CHO-K1 epithelial cells.

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Fig. 2.
Expression of myc-tagged
-tubulin in CHO-K1 cells. Cell extracts (100 µg) were
prepared from untransfected CHO-K1 cells and CHO-K1 cells expressing
myc-tagged -tubulin ( -myc) and were
subjected to SDS/polyacrylamide gel electrophoresis on 8.5%
polyacrylamide gels. After blotting onto nitrocellulose, blots were
probed with anti-myc antibody (A) or
anti- -tubulin antibody (B), as described under
"Materials and Methods." Extracts were from untransfected cells
(lanes 1) or cells stably transfected with a construct
encoding -myc (lanes 2). The
numbers on the left indicate migration of
molecular mass markers, in kDa.
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Fig. 3.
Localization of myc-tagged
-tubulin in CHO-K1 cells. Cells on glass coverslips were
processed for immunofluorescence as described under "Materials and
Methods." Untransfected CHO-K1 cells were stained with
anti- -tubulin (A) or anti-myc antibodies (B). Cells stably transfected with a construct encoding
myc-tagged -tubulin were stained with anti- -tubulin
(C) or anti-myc antibodies (D).
myc-tagged -tubulin is correctly localized to the
centrosome (n = 300).
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The Amino and Carboxyl Termini of Human
-Tubulin Are
Essential--
Constructs encoding proteins that lacked the first 10 amino-terminal amino acid residues (N
10myc) or the last 10 or 19 carboxyl-terminal residues (C
10myc or C
19myc, respectively) were
introduced into CHO-K1 cells (see Fig. 6). Stable cell lines expressing
these transgenes were generated as detected by immunoblot analysis with anti-
-tubulin and anti-myc antibodies (Fig.
4). To determine if these mutant
epitope-tagged versions of human
-tubulin were correctly localized
in CHO-K1 cells, we performed indirect immunofluorescence on cells
expressing these proteins. Staining cells with an anti-myc antibody showed that although a mutant
-tubulin protein lacking its
10 carboxyl-terminal amino acids (C
10myc) was correctly localized to
the centrosome (Fig. 5, C and
D),
-tubulin proteins lacking their 10 amino-terminal
amino acids (N
10myc) or their 19 carboxyl-terminal amino acids
(C
19myc) were not correctly localized (Fig. 5, A, B, E, and F). Stable cell lines were
generated that expressed for
-tubulin proteins that possessed more
severe amino- and carboxyl-terminal deletions. None of these mutant
proteins were correctly localized (not shown). Thus, deletion analysis
of human
-tubulin demonstrated that its 10 amino-terminal amino
acids and its 19 carboxyl-terminal amino acids were essential for
centrosomal localization.

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Fig. 4.
Expression of myc-tagged
-tubulin deletion mutants in CHO-K1 cells. Cell extracts were
prepared untransfected CHO-K1 cells and from cells expressing N 10
-tubulin-myc (N 10), C 10 -tubulin-myc
(C 10), or C 19 -tubulin-myc (C 19). Blots were prepared and processed as described in the legend to Fig. 2. Blots were
probed with anti- -tubulin antibody (A) or
anti-myc antibody (B). , endogenous
-tubulin; -myc, transgenic myc-tagged
version of -tubulin; U, untransfected cells.
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Fig. 5.
Localization of myc-tagged
-tubulin deletion mutants in CHO-K1 cells. A and
B, N 10 -tubulin-myc. C and
D, C 10 -tubulin-myc. E and
F, C 19 -tubulin-myc. Antibodies employed in
the study were anti- -tubulin (A, C, and
E) or anti-myc antibodies (B,
D, and F). Cells were processed as described in
Fig. 3. Neither N 10- -tubulin-myc nor C 19
-tubulin-myc were localized to the centrosome
(n = 300).
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The Amino-terminal Third and the 19 Carboxyl-terminal Amino Acids
of
-Tubulin Cannot Be Replaced with Those of
-Tubulin--
The
impact of deletions on
-tubulin function could have been due to the
removal of key amino acid residues directly responsible for
-tubulin-specific function, for example, by binding to specific proteins. Alternatively, these deletions may have affected folding of
the
-tubulin protein. To differentiate between these two
possibilities, we took advantage of the fact that
- and
-tubulin
show about 30% sequence similarity at the amino acid level and are
presumed to have similar overall structures (Fig.
6; Ref. 25). Yet, the amino- and
carboxyl-terminal amino acids of
- and
-tubulin show identity in
4 of 10 residues at the amino terminus and 2 of 19 residues at the
carboxyl terminus. Thus, replacing
-tubulin sequences with those of
-tubulin would not be expected to alter overall protein structure,
but if residues in the amino and carboxyl termini of
-tubulin are
essential for
-tubulin-specific function, then these substitutions
might be expected to abrogate localization of
-tubulin to the
centrosome. To assess the impact of these sequence differences on
tubulin function, we generated a series of hybrid constructs between
mouse
- and human
-tubulin and created stably transfected CHO-K1
cells expressing these hybrid proteins. We subjected these cells to
immunofluorescence with anti-myc, anti-
-tubulin, or
anti-
-tubulin antibodies. A summary of these results is shown in
Fig. 7. We determined that substituting the 10 amino-terminal amino acids of
-tubulin with those of
-tubulin (
N10
myc) did not abolish centrosomal localization.
This region of
- and
-tubulin differs in six of ten positions,
yet this difference was not sufficient to alter centrosomal
localization of
-tubulin; that is, these 10 amino-terminal residues
are not required for any detectable
-tubulin-specific function.
Conversely, substituting the amino-terminal third of
-tubulin with
those of
-tubulin (
N
myc) abolished centrosomal localization.
Similarly, replacing the carboxyl-terminal 19 amino acids of
-tubulin with those of
-tubulin (
C19
myc) resulted in a
protein incapable of localization. Thus, the amino-terminal third and
carboxyl-terminal 19 amino acids of
-tubulin are essential for a
-tubulin-specific function, namely centrosomal localization.

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Fig. 6.
Comparison of the amino acid residues of
human -tubulin (27) and mouse -tubulin (35). Residues
conserved between - and -tubulin are denoted with a
dash. An evolutionarily conserved residue (an insertion
relative to -tubulin) in the amino terminus of -tubulin is in
bold (see text). End points of amino-terminal (N 10) and
carboxyl-terminal (C 10 and C 19) -tubulin mutants mentioned in
text are shown. Residues that were exchanged between - and
-tubulin to generate hybrids in which protein thirds were exchanged
are denoted by a box.
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Fig. 7.
Summary of behavior of mutant -tubulins in
stably transfected CHO-K1 cells. White box, -tubulin
sequence; black box, -tubulin sequence. Cells were fixed
and stained with anti-myc antibody, anti- -tubulin
antibody, and, when appropriate, anti- -tubulin antibody. Whether the
myc epitope was present at centrosome is noted
(n = 300). No staining of the microtubule network was
observed. -myc is wild-type myc-tagged
-tubulin; N 10myc, C 10myc, and C 19myc were previously
described versions of -tubulin protein. N10 myc is a version of
-tubulin with its 10 amino-terminal amino acid residues replaced
with those of -tubulin; similarly C19 myc is a version of
-tubulin with its 19 carboxyl-terminal amino acids replaced with
those of -tubulin. N myc and C myc are versions of
-tubulin that have their amino- and carboxyl-terminal thirds,
respectively, replaced with those of -tubulin.
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The Amino and Carboxyl Termini of Human
-Tubulin Are Essential
for Its Incorporation into the Gammasome (
TuRC)--
After
translation,
-tubulin is incorporated into the gammasome (13, 14,
18), which becomes recruited into the centrosome, where it nucleates
filament assembly (18). Thus, the inability of our mutant
-tubulin
proteins to become appropriately localized could be caused by at least
two factors. First, the mutant
-tubulin might be defective in its
ability to be recruited into the gammasome. Alternatively, the mutant
-tubulin might retain its ability to be incorporated into the
gammasome, but the mutation abolished the ability of the gammasome to
be associated with the centrosome. To distinguish between these two
possibilities, we performed sucrose density gradient centrifugation
with cell extracts prepared from CHO-K1 cells expressing
myc-tagged wild-type
-tubulin (
-myc) or
cells expressing our amino- or carboxyl-terminal localization-defective (
N
myc or
C19
myc, respectively) hybrid proteins. Fractions from the gradients were subjected to Western blot analysis and probed
with anti-
-tubulin or anti-myc antibodies. In
untransfected CHO-K1 cells,
-tubulin was found in a 25 S complex
(Fig. 8, top panel; Ref. 14).
Conversely, in cells expressing the amino-terminal localization-defective hybrids (
N
myc), the mutant protein was not
present in the gammasome but was instead localized to the first few
fractions of the gradient, corresponding to a considerably smaller
molecule (Fig. 8, middle and bottom panels).
Furthermore, it appeared that the amino-terminal
-tubulin hybrid
mutant was cleaved into at least one other detectable form
corresponding to a carboxyl-terminal fragment of approximately 20 kDa,
only detectable with the anti-myc antibody (Fig. 8,
bottom panel). Incubation of the extract with protease
inhibitor mixtures had no effect on protein cleavage (not shown).
Similarly, when extracts prepared from CHO-K1 cells expressing
C19
myc were analyzed, mutant protein was not localized to the
gammasome (Fig. 9). Collectively, these
data suggest that the amino and carboxyl termini of
-tubulin were
essential for its incorporation into the gammasome and that the
corresponding regions of
-tubulin could not substitute for these
residues.

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Fig. 8.
Size of myc-tagged -tubulin
mutant-containing complexes in stably transfected CHO-K1 cells:
amino-terminal mutant. Sucrose gradient centrifugation was
performed ("Materials and Methods" and Ref. 14). Fractions (100 µl) were collected and assayed as described under "Materials and
Methods." Extracts were prepared from untransfected CHO-K1 cells
(top panel) and CHO-K1 cells expressing N myc, which
has the amino-terminal third of -tubulin replaced with that of
-tubulin (middle and bottom panels). Nitrocellulose filters were probed with anti-myc antibody or
anti- -tubulin antibody as denoted. Lane numbers
correspond to fraction numbers from top to bottom of the gradient.
Under the conditions of the assay, fractions 5 and 6 correspond to
approximately 25 S. N myc is not localized to the gammasome.
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Fig. 9.
Size of myc-tagged -tubulin
mutant-containing complexes in stably transfected CHO-K1 cells:
carboxyl-terminal mutant. Sucrose gradient centrifugation was
performed, and fractions were collected and assayed as described in the
legend to Fig. 8. Extracts were prepared from CHO-K1 cells expressing
C19 myc, a protein in which the 19 carboxyl-terminal amino acids
of -tubulin are replaced with those of -tubulin. Nitrocellulose
filters were probed with anti-myc antibody or
anti- -tubulin antibody as denoted. Lane numbers
correspond to fraction numbers from top to bottom of the gradient.
C19 myc is not localized to the gammasome.
C19 myc, transgenic C19 myc protein; *, transgenic
protein from which the myc tag has been clipped.
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The Amino and Carboxyl Termini of
-Tubulin Are Important for
Heterodimer Function--
Intrigued by our observations with
-tubulin, we decided to determine if amino- and carboxyl-terminal
-tubulin sequences could replace the corresponding sequences from
mouse
-tubulin. To initiate this study, we generated CHO-K1 cell
lines stably expressing wild type myc-tagged
-tubulin.
This protein was correctly integrated into the microtubule network as
determined by immunofluorescence with anti-myc antibody
(Fig. 10, A and
B). Constructs were generated that permitted expression of
mutant versions of
-tubulin in transfected cells. Proteins derived
from
-tubulin were expressed that either had its first 10 amino-terminal residues or its 20 carboxyl-terminal residues replaced
with those of
-tubulin. Stably transfected CHO-K1 cells expressing
the amino-terminal
-tubulin hybrid were generated; the transgene was
located in the cytosol (Fig. 10, C and D). These
cells proliferated readily and had no apparent phenotype in that cells
were indistunguishable in size, shape, and doubling time to
untransfected cells. An additional
-tubulin mutant was generated
that possessed a proline insertion between amino acid residues one and
two (see Fig. 6, bold letter). This residue is conserved in
-tubulin (14). This mutant protein also could not integrate into the
filament network (not shown). Collectively, these results suggest that
the 10 amino-terminal residues of
-tubulin are essential for its
appropriate subcellular localization.

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Fig. 10.
Localization of myc-tagged
mutant -tubulin proteins in transfected CHO-K1 cells. Cells
were processed for immunofluorescent staining as described under
"Materials and Methods." Anti-myc antibody and
anti- -tubulin antibodies were used as denoted on cells transfected
with wild-type myc-tagged -tubulin (A and
B), -tubulin that had its 10 amino-terminal amino acids
replaced with those of -tubulin ( N10 myc) (C and
D), and -tubulin that had its 19 carboxyl-terminal amino
acids replaced with those of -tubulin ( C19 myc) (E
and F). Cells were stained with anti- -tubulin (A, C, and E) or anti-myc
antibodies (B, D, and F). Whereas
myc-tagged -tubulin was correctly localized to the
microtubule filament network (n = 300), neither
N10 myc (n = 300) nor C19 myc
(n = 42) was appropriately localized to the microtubule
network but was instead dispersed throughout the cytosol.
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In contrast to mutations at the amino terminus of
-tubulin, no
stably transfected CHO-K1 cells expressing a hybrid
-tubulin protein
that had its carboxyl-terminal 20 amino acids replaced with those of
-tubulin could be isolated. Thus, we hypothesized that expression of
the mutant
-tubulin protein caused lethality. To test this notion,
we transiently transfected CHO-K1 cells with the appropriate DNA
construct and examined these cells for expression and subcellular
localization of the carboxyl-terminal
-tubulin hybrid protein.
Transient transfection of CHO-K1 cells with a construct encoding for
wild- type myc-tagged
-tubulin verified that the
transfected protein correctly localized to the microtubule filament
network. However, when the carboxyl-terminal
-tubulin hybrid was
transfected into CHO-K1 cells, no filament network could be detected
with either anti-
-tubulin or anti-myc antibodies;
-tubulin protein was dispersed throughout the cell. Furthermore, some transfected cells expressing the mutant protein appeared round
(Fig. 10, E and F). Inspection, before fixation,
of the tissue culture dish containing transfected cells showed an
abnormally large number of rounded cells that were readily detached
from the plate upon agitation.
The Amino Terminus of Mouse
-Tubulin Is Necessary for
Incorporation of the Heterodimer into the Microtubule
Network--
Immediately after translation,
-tubulin must be
properly folded and incorporated into a distinct soluble form, the 7 S
- and
-heterodimer. Thus, the inability of the
-tubulin mutant hybrid proteins to integrate into the filament network could be because
the mutant proteins are incapable of heterodimerizing with
-tubulin.
Alternatively, the
-tubulin mutant hybrid proteins might be capable
of heterodimerization, but these mutant heterodimers might be incapable
of normal function. To distinguish between these two possibilities,
cell extracts were prepared from untransfected CHO-K1 cells and CHO-K1
cells expressing the amino-terminal
-tubulin mutant protein
(
N10
myc). Extracts were prepared and separated by size over a gel
filtration column. Fractions were collected, concentrated, and
subjected to Western blot analysis. Not surprisingly, when extracts
from untransfected CHO-K1 cells were used in our assay, we found that
endogenous
-tubulin behaved as a heterodimer, eluting as a molecular
mass of around 100 kDa (Fig. 11,
top panel). In addition, when extract from CHO-K1 cells
stably expressing the amino-terminal
-tubulin mutant (
N10
myc)
were passed over our sizing column,
N10
myc also behaved as a
heterodimer (Fig. 11, bottom three panels). These results
suggest that although replacing the 10 amino-terminal residues of
-tubulin with those of
-tubulin did not abrogate its ability to
heterodimerize with
-tubulin, the heterodimer bearing the mutation
was not able to effectively incorporate into the microtubule filament
network.

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Fig. 11.
Size of amino-terminal
myc-tagged mutant -tubulin ( N10 myc) containing
complexes in CHO-K1 cells. Gel filtration was performed as
described under "Materials and Methods." Fractions (0.5 ml) were
collected, concentrated, and subjected to SDS/polyacrylamide gel
electrophoresis on 8.5% polyacrylamide gels. After blotting on
nitrocellulose, filters were probed with anti-myc,
anti- -tubulin, or anti- -tubulin antibodies as noted. Extracts
were prepared from untransfected CHO-K1 cells (top panel)
and cells expressing -tubulin that had its 10 amino-terminal amino
acids replaced with those of -tubulin (three bottom
panels). Sizes of molecular mass standards run with each column
are shown on top. Arrows denote the transgenic,
myc-tagged protein. N10 myc behaved as a
heterodimer.
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That permanent cell lines expressing the carboxyl-terminal
-tubulin
hybrid were not isolated and that cells transiently expressing the
mutant protein showed collapsed filaments and exhibited the characteristics of dying cells suggested that the mutant tubulin
C19
myc heterodimerized with
-tubulin and integrated into the filament network, where it exerted its effect in a dominant-negative fashion. Alternatively, the mutant protein could associate with itself
or other proteins, such as
-tubulin, to form an insoluble aggregate
in the cell. However, when we passed extracts containing the mutant
protein over our sizing column, we could not clearly and conclusively
distinguish between these two possibilities. Collectively, our results
with
-tubulin suggest that relatively minor alterations in its amino
or carboxyl termini profoundly affect activity of
-tubulin,
abolishing its ability to become properly incorporated into the
microtubule filament.
 |
DISCUSSION |
Microtubules are essential for many cell activities; for example,
they segregate sister chromatids to opposite ends of the cell and
distribute mRNAs and organelles to particular subcellular locales.
Essential to their activity is their inherent polarity, with growing,
plus ends emanating from their anchored, centrosome-associated minus
end. Three tubulins are essential for microtubule polymerization:
-,
-, and
-tubulin.
- and
-tubulin form a heterodimer that is
the building block of the microtubule filament, whereas
-tubulin can
be recruited to the centrosome, where it is essential for microtubule
nucleation (5, 8, 9, 11, 14, 16). The molecular basis for microtubule
polymerization has been the subject of much research.
Although much recent work examined how the filament grows (for a
review, see Ref. 29), relatively little is known about the structural
requirements the heterodimer has for its proper integration and
recruitment into the microtubule filament. In this report, we discover
that two relatively small domains present in the amino and carboxyl
termini of
-tubulin are necessary for heterodimer function. First,
our data show that the 10 amino-terminal residues are essential for
recruitment of the heterodimer into the filament network; a single
amino acid insertion into this region is sufficient to abolish
heterodimer incorporation into the microtubule filament network. This
observation is intriguing in light of the result that beads coated with
GTP bound to the plus end of the microtubule (30). Because
-tubulin,
but not
-tubulin, has an exchangeable GTP (20), it was concluded
that
-tubulin is present at the minus end of the filament; that is, it is believed to interact directly with
-tubulin. Recently, other
observations have confirmed that
-tubulin is present on the minus
end of the filament (31, 32, 33). Taken together with these studies,
our results suggest that the amino terminus of
-tubulin promotes
incorporation of the
-
-heterodimer into the filament by
interacting with either
-tubulin at the centrosome or with
-tubulin at the free, plus end of the microtubule.
Second, our data suggest that the 20 carboxyl-terminal residues of
-tubulin could be essential for maintenance of the microtubule filament. Replacement of these residues with those of
-tubulin resulted in collapse of the microtubule network. Transfected cells expressing the mutant protein became rounded and detached from the
tissue culture plate. We hypothesize that the role of the 20 carboxyl-terminal amino acid residues is to interact with adjacent protofilaments to promote microtubule filament polymerization or
stability. The recent determination that, in a filament, adjacent
-tubulin subunits are adjacent to one another (24) suggests that
these residues are essential for lateral associations between adjacent
-tubulin subunits. An alternative explanation of our data concerning
the carboxyl-terminal
-tubulin mutant is that the mutant protein
aggregated in the cytosol with itself or with other proteins to form a
particle of similar size to the heterodimer and that this product is
toxic to the cell. In any event, our data show that mutating the
carboxyl terminus of
-tubulin abolishes its proper function.
In cells, the gammasome complex is recruited to the centrosome;
-tubulin must be present at centrosomes that are capable of
undergoing microtubule synthesis. However,
-tubulin is not an
obligate centrosomal component (14, 15). Thus, for
-tubulin to
nucleate filament assembly, it must first be integrated into the
gammasome and then be incorporated into the centrosome. How
-tubulin
interacts with other proteins to exert its function is unknown;
however, proteins necessary for
-tubulin function are being
identified and characterized (19, 34).
Although several
-tubulin mutants have been isolated, their
phenotype has only been characterized at a gross level, namely by their
inability to nucleate filament assembly (8, 9). Until now, regions of
-tubulin necessary for particular biochemical interactions have not
been characterized. In this report, we determined that deleting either
the 10 amino-terminal or the 19 carboxyl-terminal amino acids of
-tubulin disrupted its subcellular localization to the gammasome.
Similarly, replacing the carboxyl-terminal 19 amino acids of
-tubulin with those of
-tubulin prevented localization to the
gammasome. This result is not surprising given the large sequence
divergence between the two proteins. Conversely, replacing the 10 amino-terminal amino acids of
-tubulin with those of
-tubulin had
no effect on
-tubulin localization. These results suggest that the
amino termini of
- and
-tubulin may interact with similar proteins. However, substituting the amino-terminal third of
-tubulin with that of
-tubulin abolished localization of
-tubulin to the
centrosome. Thus, replacing the amino-terminal third of
-tubulin or
its carboxyl-terminal 10 residues with those of
-tubulin abolished the ability of
-tubulin to be incorporated into gammasome. These results suggest that these regions are essential for a specific
-tubulin function, namely its recruitment to the gammasome. In fact,
it may be that regions necessary for the centrosomal localization of
-tubulin are spread out over the entire protein.
Previously, it was assumed that the high degree of sequence
conservation at the amino-terminal end of
-tubulin and
-tubulin meant that these regions of the tubulin proteins contained information necessary for direct interaction between
- and
-tubulin (25). Conversely, the high degree of sequence divergence between
- and
-tubulins at their carboxyl termini was taken as an indication that
this region contained tubulin-type-specific information (25). However,
our results suggest that information necessary for
- and
-tubulin-specific function is located at both termini and perhaps
throughout the length of these proteins, although the sequence
requirements seem to be more strict for
-tubulin than for
-tubulin. That is, the high degree of sequence conservation within
any particular tubulin gene is an indication of the high degree of
specialization resident in each tubulin protein. As further mutations
in tubulin genes are isolated and subjected to cytological and
biochemical analyses and the corresponding cellular proteins that
interact with these domains are characterized, new insights should be
forthcoming into the molecular mechanism underlying microtubule
initiation, growth, polarity establishment, and function in eukaryotic
cells.