Expression of Amino- and Carboxyl-terminal gamma - and alpha -Tubulin Mutants in Cultured Epithelial Cells*

Andrew LeaskDagger § and Tim Stearns§

From Dagger  FibroGen, Inc., South San Francisco, California 94080-6902 and the § Department of Biological Sciences, Stanford University, Stanford, California 94030-5020

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Three distinct tubulin proteins are essential for microtubule function: alpha -, beta -, and gamma -tubulin. After translation, alpha - and beta -tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, gamma -tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian alpha - and gamma -tubulin necessary for their function by creating mutant mammalian alpha - and gamma -tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxyl-terminal epitope tag had no effect on the subcellular localization of either alpha - and gamma -tubulin. Second, we found that both the amino and carboxyl termini of gamma -tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of alpha -tubulin were necessary for incorporation of the alpha -beta -tubulin heterodimer into the microtubule filament network. In general, alpha -tubulin sequences could not replace those of gamma -tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of alpha - and gamma -tubulin and perhaps regions throughout these proteins were necessary for their specific functions.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha - and beta -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 alpha - and beta -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, gamma -tubulin (3, 4). This protein is evolutionarily conserved, occurring in animals, insects, plants, and fungi (3, 5-13). gamma -Tubulin is not an obligate centrosomal component (14, 15); yet recruitment of gamma -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 gamma -tubulin are evolutionarily conserved and that gamma -tubulin is essential for microtubule nucleation suggest a universal mechanism in which gamma -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) gamma -tubulin is always found as a large 25 S, ring-like complex (termed a gammasome or gamma TuRC) that nucleates microtubule assembly when recruited to the centrosome (13, 17, 18). That gamma -tubulin is always found in the gammasome has led to the inference that gamma -tubulin must interact with other gammasomal proteins to exert its function. Although some proteins that might aid gamma -tubulin in its role have been identified, no direct evidence has been provided proving their function. For example, the gammasome includes alpha - and beta -tubulin (13). Also, an additional protein that may interact with gamma -tubulin at the centrosome has been recently identified (19). However, exactly how gamma -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 alpha - and beta -heterodimers are added to the free end of two-dimensional sheets whose lateral ends eventually circularize to form a cylinder (20) such that alpha -tubulin subunits are adjacent to alpha -tubulin subunits of another protofilament (21-24). However, exactly how the alpha -beta -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 alpha -, beta -, and gamma -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 alpha - and gamma -tubulin in their subcellular localizations and microtubule filament formation. To address this issue, we expressed epitope-tagged alpha - and gamma -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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma -tubulin (XGC1-4, raised against a region in the carboxyl terminus of Xenopus gamma -tubulin; Ref. 3); anti-alpha -tubulin (YL1/2, raised against the entire rat alpha -tubulin protein; Sera Labs); and anti-beta -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-gamma -tubulin (XGC1-4; Ref. 14); anti-alpha -tubulin (YL1/2); anti-beta -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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

myc-tagged Human gamma -Tubulin Is Correctly Localized to the Centrosome-- Previously, we had demonstrated the importance of gamma -tubulin in initiating microtubule assembly at centrosomes (14). To assess how gamma -tubulin is selected to be localized to the centrosome, we subcloned full-length human gamma -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 gamma - and mouse alpha -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.

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 gamma -tubulin was a necessary prerequisite for studying transfected mutant protein because transiently transfected gamma -tubulin was dispersed throughout the cytoplasm. CHO-K1 epithelial cells were transfected with a construct encoding full-length myc-tagged gamma -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-gamma -tubulin antibodies. Anti-gamma -tubulin antibody detected the endogenous gamma -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 gamma -tubulin was detected with both anti-gamma -tubulin (Fig. 2, lane 2) and anti-myc antibody (Fig. 2, lane 2), verifying expression of the transgenic protein. The gamma -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-gamma -tubulin staining of untransfected cells (Fig. 3, A-D). Because epitope-tagged gamma -tubulin was correctly localized to the centrosome of stably transfected CHO-K1 cells, we decided to delineate domains of human gamma -tubulin essential for its subcellular localization by introducing deletions into the amino and carboxyl termini of gamma -tubulin and assaying their behavior in stably transfected CHO-K1 epithelial cells.


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Fig. 2.   Expression of myc-tagged gamma -tubulin in CHO-K1 cells. Cell extracts (100 µg) were prepared from untransfected CHO-K1 cells and CHO-K1 cells expressing myc-tagged gamma -tubulin (gamma -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-gamma -tubulin antibody (B), as described under "Materials and Methods." Extracts were from untransfected cells (lanes 1) or cells stably transfected with a construct encoding gamma -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 gamma -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-gamma -tubulin (A) or anti-myc antibodies (B). Cells stably transfected with a construct encoding myc-tagged gamma -tubulin were stained with anti-gamma -tubulin (C) or anti-myc antibodies (D). myc-tagged gamma -tubulin is correctly localized to the centrosome (n = 300).

The Amino and Carboxyl Termini of Human gamma -Tubulin Are Essential-- Constructs encoding proteins that lacked the first 10 amino-terminal amino acid residues (NDelta 10myc) or the last 10 or 19 carboxyl-terminal residues (CDelta 10myc or CDelta 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-gamma -tubulin and anti-myc antibodies (Fig. 4). To determine if these mutant epitope-tagged versions of human gamma -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 gamma -tubulin protein lacking its 10 carboxyl-terminal amino acids (CDelta 10myc) was correctly localized to the centrosome (Fig. 5, C and D), gamma -tubulin proteins lacking their 10 amino-terminal amino acids (NDelta 10myc) or their 19 carboxyl-terminal amino acids (CDelta 19myc) were not correctly localized (Fig. 5, A, B, E, and F). Stable cell lines were generated that expressed for gamma -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 gamma -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 gamma -tubulin deletion mutants in CHO-K1 cells. Cell extracts were prepared untransfected CHO-K1 cells and from cells expressing NDelta 10 gamma -tubulin-myc (NDelta 10), CDelta 10 gamma -tubulin-myc (CDelta 10), or CDelta 19 gamma -tubulin-myc (CDelta 19). Blots were prepared and processed as described in the legend to Fig. 2. Blots were probed with anti-gamma -tubulin antibody (A) or anti-myc antibody (B). gamma , endogenous gamma -tubulin; gamma -myc, transgenic myc-tagged version of gamma -tubulin; U, untransfected cells.


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Fig. 5.   Localization of myc-tagged gamma -tubulin deletion mutants in CHO-K1 cells. A and B, NDelta 10 gamma -tubulin-myc. C and D, CDelta 10 gamma -tubulin-myc. E and F, CDelta 19 gamma -tubulin-myc. Antibodies employed in the study were anti-gamma -tubulin (A, C, and E) or anti-myc antibodies (B, D, and F). Cells were processed as described in Fig. 3. Neither NDelta 10-gamma -tubulin-myc nor CDelta 19 gamma -tubulin-myc were localized to the centrosome (n = 300).

The Amino-terminal Third and the 19 Carboxyl-terminal Amino Acids of gamma -Tubulin Cannot Be Replaced with Those of alpha -Tubulin-- The impact of deletions on gamma -tubulin function could have been due to the removal of key amino acid residues directly responsible for gamma -tubulin-specific function, for example, by binding to specific proteins. Alternatively, these deletions may have affected folding of the gamma -tubulin protein. To differentiate between these two possibilities, we took advantage of the fact that alpha - and gamma -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 alpha - and gamma -tubulin show identity in 4 of 10 residues at the amino terminus and 2 of 19 residues at the carboxyl terminus. Thus, replacing gamma -tubulin sequences with those of alpha -tubulin would not be expected to alter overall protein structure, but if residues in the amino and carboxyl termini of gamma -tubulin are essential for gamma -tubulin-specific function, then these substitutions might be expected to abrogate localization of gamma -tubulin to the centrosome. To assess the impact of these sequence differences on tubulin function, we generated a series of hybrid constructs between mouse alpha - and human gamma -tubulin and created stably transfected CHO-K1 cells expressing these hybrid proteins. We subjected these cells to immunofluorescence with anti-myc, anti-alpha -tubulin, or anti-gamma -tubulin antibodies. A summary of these results is shown in Fig. 7. We determined that substituting the 10 amino-terminal amino acids of gamma -tubulin with those of alpha -tubulin (gamma N10alpha myc) did not abolish centrosomal localization. This region of alpha - and gamma -tubulin differs in six of ten positions, yet this difference was not sufficient to alter centrosomal localization of gamma -tubulin; that is, these 10 amino-terminal residues are not required for any detectable gamma -tubulin-specific function. Conversely, substituting the amino-terminal third of gamma -tubulin with those of alpha -tubulin (gamma Nalpha myc) abolished centrosomal localization. Similarly, replacing the carboxyl-terminal 19 amino acids of gamma -tubulin with those of alpha -tubulin (gamma C19alpha myc) resulted in a protein incapable of localization. Thus, the amino-terminal third and carboxyl-terminal 19 amino acids of gamma -tubulin are essential for a gamma -tubulin-specific function, namely centrosomal localization.


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Fig. 6.   Comparison of the amino acid residues of human gamma -tubulin (27) and mouse alpha -tubulin (35). Residues conserved between gamma - and alpha -tubulin are denoted with a dash. An evolutionarily conserved residue (an insertion relative to alpha -tubulin) in the amino terminus of gamma -tubulin is in bold (see text). End points of amino-terminal (NDelta 10) and carboxyl-terminal (CDelta 10 and CDelta 19) gamma -tubulin mutants mentioned in text are shown. Residues that were exchanged between alpha - and gamma -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 gamma -tubulins in stably transfected CHO-K1 cells. White box, gamma -tubulin sequence; black box, alpha -tubulin sequence. Cells were fixed and stained with anti-myc antibody, anti-gamma -tubulin antibody, and, when appropriate, anti-alpha -tubulin antibody. Whether the myc epitope was present at centrosome is noted (n = 300). No staining of the microtubule network was observed. gamma -myc is wild-type myc-tagged gamma -tubulin; NDelta 10myc, CDelta 10myc, and CDelta 19myc were previously described versions of gamma -tubulin protein. gamma N10alpha myc is a version of gamma -tubulin with its 10 amino-terminal amino acid residues replaced with those of alpha -tubulin; similarly gamma C19alpha myc is a version of gamma -tubulin with its 19 carboxyl-terminal amino acids replaced with those of alpha -tubulin. gamma Nalpha myc and gamma Calpha myc are versions of gamma -tubulin that have their amino- and carboxyl-terminal thirds, respectively, replaced with those of alpha -tubulin.

The Amino and Carboxyl Termini of Human gamma -Tubulin Are Essential for Its Incorporation into the Gammasome (gamma TuRC)-- After translation, gamma -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 gamma -tubulin proteins to become appropriately localized could be caused by at least two factors. First, the mutant gamma -tubulin might be defective in its ability to be recruited into the gammasome. Alternatively, the mutant gamma -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 gamma -tubulin (gamma -myc) or cells expressing our amino- or carboxyl-terminal localization-defective (gamma Nalpha myc or gamma C19alpha myc, respectively) hybrid proteins. Fractions from the gradients were subjected to Western blot analysis and probed with anti-gamma -tubulin or anti-myc antibodies. In untransfected CHO-K1 cells, gamma -tubulin was found in a 25 S complex (Fig. 8, top panel; Ref. 14). Conversely, in cells expressing the amino-terminal localization-defective hybrids (gamma Nalpha 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 gamma -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 gamma C19alpha myc were analyzed, mutant protein was not localized to the gammasome (Fig. 9). Collectively, these data suggest that the amino and carboxyl termini of gamma -tubulin were essential for its incorporation into the gammasome and that the corresponding regions of alpha -tubulin could not substitute for these residues.


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Fig. 8.   Size of myc-tagged gamma -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 gamma Nalpha myc, which has the amino-terminal third of gamma -tubulin replaced with that of alpha -tubulin (middle and bottom panels). Nitrocellulose filters were probed with anti-myc antibody or anti-gamma -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. gamma Nalpha myc is not localized to the gammasome.


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Fig. 9.   Size of myc-tagged gamma -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 gamma C19alpha myc, a protein in which the 19 carboxyl-terminal amino acids of gamma -tubulin are replaced with those of alpha -tubulin. Nitrocellulose filters were probed with anti-myc antibody or anti-gamma -tubulin antibody as denoted. Lane numbers correspond to fraction numbers from top to bottom of the gradient. gamma C19alpha myc is not localized to the gammasome. gamma C19alpha myc, transgenic gamma C19alpha myc protein; *, transgenic protein from which the myc tag has been clipped.

The Amino and Carboxyl Termini of alpha -Tubulin Are Important for Heterodimer Function-- Intrigued by our observations with gamma -tubulin, we decided to determine if amino- and carboxyl-terminal gamma -tubulin sequences could replace the corresponding sequences from mouse alpha -tubulin. To initiate this study, we generated CHO-K1 cell lines stably expressing wild type myc-tagged alpha -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 alpha -tubulin in transfected cells. Proteins derived from alpha -tubulin were expressed that either had its first 10 amino-terminal residues or its 20 carboxyl-terminal residues replaced with those of gamma -tubulin. Stably transfected CHO-K1 cells expressing the amino-terminal alpha -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 alpha -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 gamma -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 alpha -tubulin are essential for its appropriate subcellular localization.


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Fig. 10.   Localization of myc-tagged mutant alpha -tubulin proteins in transfected CHO-K1 cells. Cells were processed for immunofluorescent staining as described under "Materials and Methods." Anti-myc antibody and anti-alpha -tubulin antibodies were used as denoted on cells transfected with wild-type myc-tagged alpha -tubulin (A and B), alpha -tubulin that had its 10 amino-terminal amino acids replaced with those of gamma -tubulin (alpha N10gamma myc) (C and D), and alpha -tubulin that had its 19 carboxyl-terminal amino acids replaced with those of gamma -tubulin (alpha C19gamma myc) (E and F). Cells were stained with anti-gamma -tubulin (A, C, and E) or anti-myc antibodies (B, D, and F). Whereas myc-tagged alpha -tubulin was correctly localized to the microtubule filament network (n = 300), neither alpha N10gamma myc (n = 300) nor alpha C19gamma myc (n = 42) was appropriately localized to the microtubule network but was instead dispersed throughout the cytosol.

In contrast to mutations at the amino terminus of alpha -tubulin, no stably transfected CHO-K1 cells expressing a hybrid alpha -tubulin protein that had its carboxyl-terminal 20 amino acids replaced with those of gamma -tubulin could be isolated. Thus, we hypothesized that expression of the mutant alpha -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 alpha -tubulin hybrid protein. Transient transfection of CHO-K1 cells with a construct encoding for wild- type myc-tagged alpha -tubulin verified that the transfected protein correctly localized to the microtubule filament network. However, when the carboxyl-terminal alpha -tubulin hybrid was transfected into CHO-K1 cells, no filament network could be detected with either anti-alpha -tubulin or anti-myc antibodies; alpha -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 alpha -Tubulin Is Necessary for Incorporation of the Heterodimer into the Microtubule Network-- Immediately after translation, alpha -tubulin must be properly folded and incorporated into a distinct soluble form, the 7 S alpha - and beta -heterodimer. Thus, the inability of the alpha -tubulin mutant hybrid proteins to integrate into the filament network could be because the mutant proteins are incapable of heterodimerizing with beta -tubulin. Alternatively, the alpha -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 alpha -tubulin mutant protein (alpha N10gamma 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 alpha -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 alpha -tubulin mutant (alpha N10gamma myc) were passed over our sizing column, alpha N10gamma myc also behaved as a heterodimer (Fig. 11, bottom three panels). These results suggest that although replacing the 10 amino-terminal residues of alpha -tubulin with those of gamma -tubulin did not abrogate its ability to heterodimerize with beta -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 alpha -tubulin (alpha N10gamma 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-beta -tubulin, or anti-alpha -tubulin antibodies as noted. Extracts were prepared from untransfected CHO-K1 cells (top panel) and cells expressing alpha -tubulin that had its 10 amino-terminal amino acids replaced with those of gamma -tubulin (three bottom panels). Sizes of molecular mass standards run with each column are shown on top. Arrows denote the transgenic, myc-tagged protein. alpha N10gamma myc behaved as a heterodimer.

That permanent cell lines expressing the carboxyl-terminal alpha -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 alpha C19gamma myc heterodimerized with beta -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 beta -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 alpha -tubulin suggest that relatively minor alterations in its amino or carboxyl termini profoundly affect activity of alpha -tubulin, abolishing its ability to become properly incorporated into the microtubule filament.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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: alpha -, beta -, and gamma -tubulin. alpha - and beta -tubulin form a heterodimer that is the building block of the microtubule filament, whereas gamma -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 alpha -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 beta -tubulin, but not alpha -tubulin, has an exchangeable GTP (20), it was concluded that alpha -tubulin is present at the minus end of the filament; that is, it is believed to interact directly with gamma -tubulin. Recently, other observations have confirmed that alpha -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 alpha -tubulin promotes incorporation of the alpha -beta -heterodimer into the filament by interacting with either gamma -tubulin at the centrosome or with beta -tubulin at the free, plus end of the microtubule.

Second, our data suggest that the 20 carboxyl-terminal residues of alpha -tubulin could be essential for maintenance of the microtubule filament. Replacement of these residues with those of gamma -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 alpha -tubulin subunits are adjacent to one another (24) suggests that these residues are essential for lateral associations between adjacent alpha -tubulin subunits. An alternative explanation of our data concerning the carboxyl-terminal alpha -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 alpha -tubulin abolishes its proper function.

In cells, the gammasome complex is recruited to the centrosome; gamma -tubulin must be present at centrosomes that are capable of undergoing microtubule synthesis. However, gamma -tubulin is not an obligate centrosomal component (14, 15). Thus, for gamma -tubulin to nucleate filament assembly, it must first be integrated into the gammasome and then be incorporated into the centrosome. How gamma -tubulin interacts with other proteins to exert its function is unknown; however, proteins necessary for gamma -tubulin function are being identified and characterized (19, 34).

Although several gamma -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 gamma -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 gamma -tubulin disrupted its subcellular localization to the gammasome. Similarly, replacing the carboxyl-terminal 19 amino acids of gamma -tubulin with those of alpha -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 gamma -tubulin with those of alpha -tubulin had no effect on gamma -tubulin localization. These results suggest that the amino termini of alpha - and gamma -tubulin may interact with similar proteins. However, substituting the amino-terminal third of gamma -tubulin with that of alpha -tubulin abolished localization of gamma -tubulin to the centrosome. Thus, replacing the amino-terminal third of gamma -tubulin or its carboxyl-terminal 10 residues with those of alpha -tubulin abolished the ability of gamma -tubulin to be incorporated into gammasome. These results suggest that these regions are essential for a specific gamma -tubulin function, namely its recruitment to the gammasome. In fact, it may be that regions necessary for the centrosomal localization of gamma -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 alpha -tubulin and gamma -tubulin meant that these regions of the tubulin proteins contained information necessary for direct interaction between alpha - and gamma -tubulin (25). Conversely, the high degree of sequence divergence between alpha - and gamma -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 alpha - and gamma -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 alpha -tubulin than for gamma -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.

    ACKNOWLEDGEMENT

We thank B. Oakley (Ohio State University, Columbus, OH) for human gamma -tubulin cDNA.

    FOOTNOTES

* This work was supported by funds from the Medical Research Council of Canada (to A. L.) and the National Institutes of Health (to T. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: FibroGen, Inc., 260 Littlefield Ave., S. San Francisco, CA 94080-6902. Tel.: 650-635-1500; Fax: 650-635-1512; E-mail: aleask{at}fibrogen.com.

1 The abbreviations used are: MTOC, microtubule organizing center; CHO, Chinese hamster ovary; CMV, cytomegalovirus; PBS, phosphate-buffered saline.

2 T. Stearns, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Pickett-Heaps, J. D. (1969) Cytobios 3, 257-280
  2. Bergen, L. G., and Borisy, G. G. (1980) J. Cell Biol. 84, 141-150[Abstract]
  3. Stearns, T., Evans, L., and Kirschner, M. (1991) Cell 65, 825-836[Medline] [Order article via Infotrieve]
  4. Moudjou, M., Bordes, N., Paintrand, M., and Bornens, M. (1996) J. Cell Sci. 109, 875-887[Abstract/Free Full Text]
  5. Horio, T., Uzawa, S., Jung, M. K., Oakley, B. R., Tanaka, K., Yanagida, M. (1991) J. Cell Sci. 99, 693-700[Abstract]
  6. Liu, B., Joshi, H. C., Wilson, T. J., Silflow, C. D., Palevitz, B. A., Snustad, D. P. (1990) Plant Cell 6, 303-314[Abstract/Free Full Text]
  7. Lopez, I., Khan, S., Sevik, M., Cande, W. Z., Hussey, H. J. (1995) Plant Physiol. 107, 309-310[Free Full Text]
  8. Marschall, L. G., Jeng, R. L., Mulholland, J., and Stearns, T. (1996) J. Cell Biol. 134, 443-454[Abstract]
  9. Oakley, B. R., Oakley, C. E., Yoon, Y., and Jung, M. K. (1990) Cell 61, 1289-1301[Medline] [Order article via Infotrieve]
  10. Oakley, C. E., and Oakley, B. R. (1989) Nature 338, 662-664[CrossRef][Medline] [Order article via Infotrieve]
  11. Sobel, S. G., and Snyder, M. (1995) J. Cell Biol. 131, 1775-1788[Abstract]
  12. Spang, A., Courtney, I., Fackler, U., Matzner, M., and Schiebel, E. (1993) J. Cell Biol. 123, 405-416[Abstract]
  13. Zheng, Y., Wong, M. L., Alberts, B., and Mitchison, T. (1995) Nature 378, 578-583[CrossRef][Medline] [Order article via Infotrieve]
  14. Stearns, T., and Kirschner, M. (1994) Cell 76, 623-638[Medline] [Order article via Infotrieve]
  15. Leask, A., Obrietan, K., and Stearns, T. (1997) Neurosci. Lett. 229, 17-20[CrossRef][Medline] [Order article via Infotrieve]
  16. Joshi, H. C., Monica, J. P., McNamara, L., and Cleveland, D. W. (1992) Nature 356, 80-83[CrossRef][Medline] [Order article via Infotrieve]
  17. Raff, J. W., Kellogg, D. R., and Alberts, B. M. (1993) J. Cell Biol. 121, 823-835[Abstract]
  18. Moritz, M., Braunfeld, M. B., Sedat, J. W., Alberts, B., Agard, D. (1995) Nature 378, 638-640[CrossRef][Medline] [Order article via Infotrieve]
  19. Geissler, S., Pereira, G., Spang, A., Knop, M., Soues, S., Kilmartin, J., and Schiebel, E. (1996) EMBO J. 15, 3899-3911[Abstract]
  20. Mandelkow, E.-M., Mandelkow, E., and Milligan, R. A. (1991) J. Cell Biol. 114, 977-991[Abstract]
  21. Amos, L. A., and Klug, A. (1974) J. Cell Sci. 14, 523-549[Medline] [Order article via Infotrieve]
  22. Wade, R. H., Horowitz, R., and Milligan, R. A. (1995) Proteins Struct. Funct. Genet. 23, 502-509 [Medline] [Order article via Infotrieve]
  23. Kikkawa, M., Ishakawa, T., Wakabayashi, T., and Hirokawa, N. (1995) Nature 376, 271-274[CrossRef][Medline] [Order article via Infotrieve]
  24. Sosa, H., and Milligan, R. A. (1996) J. Mol. Biol. 260, 742-755
  25. Burns, R. G. (1995) J. Cell Sci. 108, 2123-2130[Free Full Text]
  26. Kube-Granderath, E., and Schliwa, M. (1997) Eur. J. Cell Biol. 72, 287-296[Medline] [Order article via Infotrieve]
  27. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1987) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  28. Horio, T., and Oakley, B. R. (1994) J. Cell Biol. 126, 1465-1473[Abstract]
  29. Wade, R. H., and Hyman, A. A. (1997) Curr. Opin. Cell Biol. 9, 12-17[CrossRef][Medline] [Order article via Infotrieve]
  30. Mitchison, T. J. (1993) Science 261, 1044-1047[Medline] [Order article via Infotrieve]
  31. Kirschner, K., and Mandelkow, E.-M. (1985) EMBO J. 4, 2397-2402[Abstract]
  32. Hirose, K., Fan, J., and Amos, L. A. (1995) J. Mol. Biol. 259, 329-333[CrossRef]
  33. Fan, J., Griffith, A. D., Lockhart, A., and Cross, R. A. (1996) J. Mol. Biol. 259, 325-330[CrossRef][Medline] [Order article via Infotrieve]
  34. Knop, M., Pereira, G., Geissler, S., Grein, K., and Schiebel, E. (1997) EMBO J. 16, 1550-1564[Abstract/Free Full Text]
  35. Lewis, S. A., Lee, M. G., and Cowan, N. J. (1985) J. Cell Biol. 101, 852-861[Abstract]


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