The Cofactor-Dependent Pathways for - and ß-Tubulins in Microtubule Biogenesis Are Functionally Different in Fission Yeast

Corresponding author: Takashi Toda, Laboratory of Cell Regulation, Imperial Cancer Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom., toda{at}europa.lif.icnet.uk (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The biogenesis of microtubules in the cell comprises a series of complex steps, including protein-folding reactions catalyzed by chaperonins. In addition a group of evolutionarily conserved proteins, called cofactors (A to E), is required for the production of assembly-competent -/ß-tubulin heterodimers. Using fission yeast, in which alp11⁺, alp1⁺, and alp21⁺, encoding the homologs for cofactors B, D, and E, respectively, are essential for cell viability, we have undertaken the genetic analysis of alp31⁺, the homolog of cofactor A. Gene disruption analysis shows that, unlike the three genes mentioned above, alp31⁺ is dispensable for cell growth and division. Nonetheless, detailed analysis of alp31-deleted cells demonstrates that Alp31^A is required for the maintenance of microtubule structures and, consequently, the proper control of growth polarity. alp31-deleted cells show genetic interactions with mutations in ß-tubulin, but not in -tubulin. Budding yeast cofactor A homolog RBL2 is capable of suppressing the polarity defects of alp31-deleted cells. We conclude that the cofactor-dependent biogenesis of microtubules comprises an essential and a nonessential pathway, both of which are required for microtubule integrity.

ALL eukaryotic cells utilize the microtubule cytoskeleton for a wide variety of cellular processes. Microtubules, biopolymers of - and ß-tubulins, initiate their assembly from specialized structures in the cell called microtubule-organizing centers (MTOCs), and free /ß-heterodimers are then incorporated into their plus-ends. The biogenesis of microtubules in vivo consists of a cascade of sequential reactions. After translation from mRNA, both - and ß-tubulins are captured in the cytosol by a group of chaperones, called chaperonins, which belong to a subfamily of GroEL and Hsp60 (CCT/TriC/c-cpn; ROMMELAERE et al. 1993 ; KUBOTA et al. 1994 ). Unlike actin and -tubulin, which also require the chaperonin complex for proper folding (GAO et al. 1992 ; MELKI et al. 1993 ; STERNLICHT et al. 1993 ), for the production of assembly competent -/ß-tubulin heterodimers, an additional set of proteins, called cofactors, is needed (CLEVELAND et al. 1978 ; GAO et al. 1993 , GAO et al. 1994 ). Molecular cloning of these cofactors from vertebrates has revealed that they comprise multiple proteins, consisting of A, B, C, D, and E (LLOSA et al. 1996 ; MELKI et al. 1996 ; TIAN et al. 1996 , TIAN et al. 1997 ).

It has been proposed, on the basis of mammalian in vitro reactions, that the pathways leading to correctly folded /ß-heterodimers comprise two symmetrical branches as summarized below. After release from the chaperonin complex (LEWIS et al. 1992 ; YAFFE et al. 1992 ), - and ß-tubulins initially follow distinct folding pathways; -tubulins are captured by cofactor B, while ß-tubulin is captured by cofactor A, which are subsequently replaced by cofactors E and D, respectively. The two pathways then converge with the formation of a quaternary complex (-tubulin/E and ß-tubulin/D). Finally cofactor C binds the complex and upon GTP hydrolysis assembly-competent -/ß-tubulin heterodimers are released (LEWIS et al. 1997 ).

In fission yeast as in higher eukaryotes, microtubules play a crucial role in chromosome segregation (UMESONO et al. 1983A ; TODA et al. 1984 ), distribution of the organella such as the mitochondria and Golgi apparatus (AYSCOUGH et al. 1993 ; YAFFE et al. 1996 ), and cell polarity (UMESONO et al. 1983B ; HIRAOKA et al. 1984 ; MATA and NURSE 1997 ; RADCLIFFE et al. 1998 ). In addition to chaperonins, all the cofactor homologs except for cofactor C exist in the genome and the homologs of cofactors B, D, and E (Alp11, Alp1, and Alp21/Sto1, respectively) have all been shown to be involved in microtubule biogenesis (HIRATA et al. 1998 ; RADCLIFFE et al. 1998 , RADCLIFFE et al. 1999 , RADCLIFFE et al. 2000 ; GRISHCHUK and MCINTOSH 1999 ; RADCLIFFE and TODA 2000 ). In contrast to the nonessential roles of these cofactor homologs in budding yeast (HOYT et al. 1990 , HOYT et al. 1997 ; STEARNS et al. 1990 ; URSIC and CULBERTSON 1991 ; ARCHER et al. 1995 ; TIAN et al. 1997 ), in fission yeast Alp11^B, Alp1^D, and Alp21^E are absolutely required for cell viability and the absence of any one results in the failure to maintain microtubule structures and in subsequent cell division defects (HIRATA et al. 1998 ; GRISHCHUK and MCINTOSH 1999 ; RADCLIFFE et al. 1999 ).

Results obtained from studies in yeasts are mostly in line with the pathways proposed from studies in higher systems; however, there apparently exists a significant difference. While the findings from the two systems are consistent with the idea that cofactors D and E act at the steps later than cofactor B, in contrast to the parallel roles of cofactors D and E in mammalian systems, in fission yeast it is evident from genetic analysis that Alp1^D functions downstream of Alp21^E (Alp11^B-Alp21^E-Alp1^D; RADCLIFFE et al. 1999 ; Table 1). This is in agreement with the result from budding yeast (Pac2^E-Cin1^D; HOYT et al. 1997 ). In addition a subpopulation of Alp1^D, but not Alp21^E, colocalizes with microtubules and biochemically behaves like microtubule-associated proteins (MAPS; HIRATA et al. 1998 ; GRISHCHUK and MCINTOSH 1999 ; RADCLIFFE et al. 1999 ). A further inconsistency is that alp1 and cin1 mutants clearly show compromised -tubulin function rather than defects in ß-tubulin, such as a lower level of -tubulin in these mutant cells (HOYT et al. 1990 ; RADCLIFFE et al. 1999 ; RADCLIFFE and TODA 2000 ). These results suggest that the pathways leading to heterodimer formation for - and ß-tubulins are not simply functionally symmetrical.

View this table: In this window In a new window   Table 1. S. pombe cofactor homologs involved in the -tubulin pathway

To address the in vivo role of the ß-tubulin pathway, we have undertaken the genetic study of the cofactor A homolog in fission yeast (designated alp31⁺). Cofactor A (Rbl2 in budding yeast) has been shown to act specifically in the ß-tubulin pathway in parallel with cofactor B (Alf1 in budding yeast and Alp11 in fission yeast), which functions only for -tubulin (GAO et al. 1993 ; ARCHER et al. 1995 , ARCHER et al. 1998 ; MELKI et al. 1996 ; RADCLIFFE et al. 1999 ; RADCLIFFE and TODA 2000 ). Here we show that, in contrast to the alp11⁺ gene, alp31⁺ is dispensable for cell viability. Nevertheless, Alp31 plays an important role in the maintenance of microtubule integrity and the determination of cell polarity. The analysis in fission yeast presented here highlights the functional similarities and differences between the cofactor-dependent pathways for - and ß-tubulin in microtubule biogenesis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and genetic methods:
Strains used in this study are listed in Table 2. YPD (2% dextrose, 2% polypeptone, and 1% yeast extract) and YE5S were used as rich media. Standard methods were followed as described (MORENO et al. 1991 ).

Nucleic acids preparation and manipulation:
Standard molecular biology techniques were followed as described (SAMBROOK et al. 1989 ). Enzymes were used as recommended by the suppliers (New England Biolabs, Beverly, MA). Nucleotide sequence data reported in this paper are in the DDBJ/EMBL/GenBank databases under accession no. AB029481 (alp31⁺).

Gene disruption:
The alp31⁺ gene was deleted using PCR-generated fragments (BAHLER et al. 1998 ). Dissection of asci from heterozygous diploid cells (PR21, Table 2) showed that alp31⁺ is a nonessential gene as all four spores germinated and formed colonies. Resistance for G418 segregated 2:2 and correct disruption of alp31⁺ was confirmed by PCR (PR23 and PR25).

Synthetic lethal interaction:
alp31-deleted cells (alp31, PR31) were crossed with various mutants defective in microtubule function. Mutants used included cold-sensitive (cs) or temperature-sensitive (ts) ß-tubulin mutants (KM311-201, nda3-311 or DHH1828, nda3-1828, respectively), cs 1-tubulin mutant (KM52-201, nda2-52), and ts or deleted 2-tubulin mutant (DHH1377, atb2-1377 or atb2, atb2::LEU2, respectively). After tetrad dissection, spores were allowed to germinate at 26° (ts) or 32° (cs).

Suppression of alp31 by expression of the budding yeast RBL2 gene:
A alp31 strain (PR31) was cotransformed with pREP1 and pREP2 or pREP1 and pA21A (RBL2-containing multicopy plasmids, obtained from Dr. F. Solomon's laboratory). Leu⁺Ura⁺ transformants were streaked on minimal medium and morphology was observed under phase-contrast microscopy.

Overexpression and epitope-tagging:
The entire open reading frame (ORF) of the alp31⁺ gene was cloned by PCR into pREP1 under control of the nmt1 promoter (MAUNDRELL 1990 ), yielding pREP1-alp31⁺. pREP1-alp31⁺ is functional, as it is capable of suppressing the morphological defects of alp31-deleted cells. To epitope-tag alp31⁺, GST was tagged at the C terminus of Alp31 using a PCR-based method (BAHLER et al. 1998 ). Tagging does not interfere with Alp31 function, as the cell morphology of a strain containing Alp31-GST is normal. Cells containing pREP1-alp31⁺ were grown in thiamine-free minimal medium for 24 hr and processed for immunofluorescence microscopy.

Immunochemical assays:
Antibodies used in this study were as follows: mouse monoclonal anti-Cdc2 antibody (obtained from Dr. Hiroyuki Yamano), mouse monoclonal anti-HA antibodies (16B12, BAbCO), mouse monoclonal anti--tubulin antibody (TAT-1, provided by Dr. Keith Gull), mouse monoclonal anti-ß-tubulin antibody (KMX-1, provided by Dr. Keith Gull), and rabbit polyclonal anti-GST antibody (G7781; Sigma Chemical Co., St. Louis, MO). Preparation of rabbit polyclonal anti-Alp11 antibody was described previously (RADCLIFFE et al. 1999 ). Horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (Bio-Rad Laboratories Ltd., Hercules, CA), and a chemiluminescence system (ECL; Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK) were used to detect bound antibody. Fission yeast whole cell extracts were prepared using glass beads to disrupt cells as described before (RADCLIFFE et al. 1998 ).

Gel filtration chromatography:
Gel filtration chromatography was performed on a Superose-6 column (Amersham Pharmacia Biotech Ltd.) in buffer A (20 mM Tris-HCl, pH 7.5, 20% glycerol, 0.1 mM EDTA, 1 mM mercaptoethanol, 5 mM ATP plus a cocktail of inhibitors). The column was equilibrated with two column volumes of buffer A containing 100 mM NaCl. To determine molecular weight, a parallel column was run with standards consisting of dextran (2000 kD), thyroglobulin (669 kD), -amylase (232 kD), and ovalbumin (43 kD). Fractions (50 µl each) were separated by SDS-PAGE and fractionated proteins were detected with individual antibodies.

Indirect immunofluorescence microscopy:
Cells were fixed with methanol and primary antibodies (TAT-1) were applied, followed by Cy3-conjugated sheep anti-mouse IgG (Sigma). Microtubules were viewed with a chilled video-rated CCD camera (model C5985, Hamamatsu, Japan) connected to a computer (Apple Power Macintosh G3/400, Cupertino, CA). Images were processed by use of Adobe Photoshop (version 4).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cofactor A homolog in fission yeast:
A homology search using human cofactor A as a query against the Schizosaccharomyces pombe genome database revealed that fission yeast contains one ORF that encodes a homologous protein (30% identity and 52% similarity; Fig 1A). This value is close to that between budding yeast Rbl2 and vertebrate cofactor A (29% identity and 57% similarity; ARCHER et al. 1995 ). The corresponding gene was designated alp31⁺ (altered polarity 31, as the gene is involved in growth polarity control, see below). The computer-assisted analysis of potential secondary structures predicted that Alp31 and vertebrate cofactor A contain two internal coiled-coil regions as shown in Fig 1B.

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Alp31 is a homolog of cofactor A. (A) Amino acid sequence comparison of fission yeast Alp31, human cofactor A, and budding yeast Rbl2. Amino acid residues, either identical (black) or conserved (gray), are emphasized. (B) Predicted coiled-coil structure of Alp31 and human cofactor A. Coiled-coil probability was determined by the Macstripe 2.0 program using window lengths of 21 amino acid residues.

alp31⁺ is a nonessential gene:
As a first step in addressing the cellular function of alp31⁺, gene disruption was performed. The entire ORF of one of the two chromosomal alp31⁺ genes in a diploid was deleted (PR21; Table 2) and tetrad analysis was performed. In contrast to our previous results, which demonstrated that the cofactor B, D, and E homologs encoding alp11⁺, alp1⁺, and alp21⁺, respectively, are essential for cell viability (HIRATA et al. 1998 ; RADCLIFFE et al. 1999 ), the alp31-deleted (alp31) cells were viable (Fig 2A). alp31 cells were neither ts nor cs. Their doubling time was 150 min in rich medium at 30°, whereas that of wild type was 130 min (Fig 2B), showing a modestly prolonged cell division. The absence of Alp31 did not alter sensitivity to antimicrotubular drugs (Fig 2C). However, a small population (2–5%) of alp31 cells showed either bent or branched morphology (arrowheads in Fig 2D, a and c), which has never been observed in wild-type cells (Fig 2D, Fig B and Fig D) and is indicative of growth polarity defects ascribable to compromised microtubule function (UMESONO et al. 1983B ; HIRAOKA et al. 1984 ). This result suggested that the fission yeast cofactor A homolog, although not essential, plays a role in microtubule integrity.

View larger version (30K): In this window In a new window Download PPT slide   Figure 2. alp31+ is not essential for cell viability. (A) Gene disruption of alp31+. A set of four segregants (a, b, c, and d: PR23, PR22, PR25, and PR24, respectively; Table 2) dissected from one ascus were grown on a rich YE5S plate at 29° (top) or at 36° (bottom), or on a YE5S plate containing 100 µg/ml of G418 (middle). (B) Growth curve of an alp31-deleted strain. Wild-type (PR22) or alp31 (PR23) cells were grown in rich liquid medium at 30° and cell number was measured. (C) Wild-type (WT, HM123) and alp31-deleted strains (alp31, PR26) were spotted onto rich YPD (-TBZ, left) or YPD plates containing 20 µg/ml of thiabendazole (+TBZ, right) as serial dilutions (106 cells in the top row and then diluted 10-fold in each subsequent spot below) and incubated at 30°. (D) Cell morphology of alp31-deleted mutants. Phase-contrast micrograph of cells taken from plates in A was presented. Branched cells are marked by arrowheads. Bar, 10 µm.

alp31-deleted cells have unstable microtubule structures but normal cellular levels of tubulins:
The altered morphology of alp31 cells suggests that microtubule function is somehow compromised. To examine this possibility, indirect immunofluorescence microscopy was performed using antitubulin antibody. It was found that microtubules are indeed defective in alp31. Using standard fixation conditions under which filamentous microtubules of wild-type cells were easily visualized (Fig 3A, bottom), no intact microtubules were evident, instead either short, dotted, or misoriented microtubules were observed (top). It appears that microtubules become highly unstable in the absence of Alp31^A.

View larger version (47K): In this window In a new window Download PPT slide   Figure 3. alp31-deleted cells have unstable microtubules and are sensitive to increased levels of the cofactor E homolog Alp21. (A) Defective microtubule structures in the alp31 disruptant. alp31 (top) or wild-type cells (bottom) were fixed, stained with anti-tubulin antibody (TAT-1, left), and processed for immunofluorescence microscopy. Chromosomal DNAs were stained with 4',6-diamidino-2-phenylin-dale (DAPI; right). Bar, 10 µm. (B) Tubulin levels. Cell extracts were prepared from alp31 (lane 1), wild type (lane 2), or wild type containing an empty plasmid (lane 3), or plasmid containing nmt1-alp31+ (pREP1-alp31+, lane 4, 24 hr in minimal medium). Immunoblotting was performed with anti-ß-tubulin antibody (KMX-1), anti--tubulin antibody (TAT-1), or anti-Cdc2 antibody. (C) Lethal overexpression of alp21+ in alp31 cells. alp31 cells containing the plasmids (clockwise from top) pREP1-alp21+, pREP1-alp11+, pREP1, pDB(alp1+), pREP1-alp31+, pREP1, or pREP1-alp1+ were streaked on minimal plates in the presence (left plate) or absence (middle) of thiamine and incubated at 26° for 3 days.

We showed previously that the temperature-sensitive alp1 and alp11 mutants, in which microtubules fail to assemble at the restrictive temperature, show reduced levels of -tubulin (RADCLIFFE et al. 1999 ). To examine whether a similar phenomenon occurred in alp31 cells, immunoblotting was performed against extracts prepared from this mutant. In contrast to the finding for the alp1 and alp11 mutants, the amount of either -tubulin or ß-tubulin did not alter significantly in this mutant (Fig 3B). This result is in line with the relatively mild phenotypes of a alp31 strain and supports the view that the pathway in which Alp31^A is involved plays a more minor role than Alp11^B, Alp21^E, and Alp1^D.

To further examine the effect of loss of Alp31^A, in particular in terms of the disturbance of the relative stoichiometry among cofactors, each cofactor homolog was overproduced in alp31 cells. In addition to Alp1^D and Alp11^B, which have been shown to be toxic even in a wild-type background (HIRATA et al. 1998 ; RADCLIFFE et al. 1999 ), it was found that Alp21^E, which does not interfere with the growth of wild-type cells, completely inhibits colony formation in alp31 cells (Fig 3C). Thus Alp31^A is important for maintaining viability under the conditions where the level of Alp21^E is increased. It is of note that multicopy plasmids containing nda2⁺ (1-tubulin) or nda3⁺ (ß-tubulin) were not capable of suppressing the morphological defects of alp31 cells.

Genetic interaction between alp31 deletion and mutations in genes involved in microtubule function:
To examine the interaction between Alp31 and tubulins, genetic crosses were performed between alp31 and ts or cs tubulin mutants such as cs nda2 (encoding 1-tubulin; TODA et al. 1984 ), deleted or ts atb2 (2-tubulin; ADACHI et al. 1986 ; RADCLIFFE et al. 1998 ), and ts or cs nda3 (ß-tubulin; HIRAOKA et al. 1984 ; RADCLIFFE et al. 1998 ). Strong synthetic interactions were found with both ts and cs ß-tubulin mutants. alp31 was synthetically lethal with ts nda3-1828 (Fig 4A), and in the case of cs nda3-311, double mutants were not capable of forming colonies at 26° (Fig 4B), which was permissive for an nda3-311 single mutant. On the other hand, mutants defective in -tubulin genes did not show synthetic phenotypes (Table 3). These results suggest that Alp31, although nonessential, is involved in the ß-tubulin pathway as reported for other organisms (ARCHER et al. 1995 ; LEWIS et al. 1997 ).

View larger version (62K): In this window In a new window Download PPT slide   Figure 4. Genetic interaction of alp31-deleted mutant and rescue by budding yeast RBL2. (A) Synthetic lethal interaction between alp31 deletion and ts nda3. Tetrad analyses between alp31 (PR31) and nda3-1828 (DHH1828) strains were performed and spores were germinated at 26°. Segregation patterns (TT, tetratype; PD; parental ditype; NPD, nonparental ditype) of each tetrad (numbers 1–8) are shown. Nonviable spores are all alp31nda3-1828 double mutants. (B) Synthetic interaction between alp31 deletion and cs nda3. Four different segregants (HM123, PR31, KM311-201, and TP509-1B) were streaked on rich plates and incubated at 30° (left) or 26° (middle) for 4 days. (C) Suppression of the morphological defects of alp31 by RBL2. Cell morphology of alp31 (PR31) containing vectors (pREP1 and pREP2, left) or plasmids carrying RBL2 (pREP1 and pA21A, right) is shown. Bar, 10 µm.

To examine whether cofactor A from another organism is capable of complementing alp31, multicopy plasmids containing the budding yeast RBL2 gene were introduced into alp31 cells. Compared to the altered cell morphology of transformants containing empty vectors only (Fig 4C, left), alp31 cells expressing RBL2 showed almost normal cylindrical shape (right), indicating that RBL2 suppressed the polarity defects of alp31 cells. Therefore, not only from structural similarity, but also on the basis of functional complementation, Alp31 is the fission yeast homolog of cofactor A.

Ectopic overexpression of alp31⁺ is toxic and results in abnormal microtubules:
To examine the phenotypes arising from overproduction of Alp31^A, the entire ORF of the alp31⁺ gene was inserted into plasmids containing the thiamine-repressible nmt1 promoter (pREP1-alp31⁺). It was found that alp31⁺ is deleterious to the cell as Alp31^A-overproducing cells show elongated, sometimes bent, morphology (Fig 5A). It is of note that pREP1-alp31⁺ did not perturb growth or colony formation; the abnormally elongated Alp31^A-overproducing cells could manage to divide and form colonies (Fig 5C).

View larger version (59K): In this window In a new window Download PPT slide   Figure 5. Ectopic overproduction of Alp31 results in compromised microtubule structures. (A) Cell morphology of Alp31-overproducing cells. Wild-type cells containing an empty vector (pREP1, left) or plasmid containing nmt1-alp31+ (pREP1-alp31+, right) were grown in the absence of thiamine (derepressed conditions) for 24 hr at 30°. (B) Defective microtubules in Alp31-overproducing cells. Cultures used in A were fixed and processed for immunofluorescence microscopy using anti-tubulin antibody (TAT-1, top). Chromosomal DNAs were stained with DAPI (bottom). Bar, 10 µm. (C) Toxic overproduction of Alp31 in ß-tubulin mutant. pREP1-alp31+ was introduced into wild type, alp31, or ts nda3-1828 mutants. Transformants were streaked on minimal plates in the presence (left) or absence (middle) of thiamine and incubated at 26° for 4 days.

To examine whether overproduction of Alp31^A results in defects in microtubule integrity, immunofluorescence microscopy was performed with anti-tubulin antibody. Staining of these alp31⁺-overexpressing cells with anti-tubulin antibody revealed that in the majority of cells no intact microtubules were present, instead none, shorter, or dotted microtubule structures were evident (Fig 5B). In spite of their defective microtubules, the total amount of tubulin molecules was unaltered in Alp31^A-overproducing cells (Fig 3B, lane 4). It is concluded that an excess amount of Alp31^A disrupts normal microtubules, leading to growth polarity defects without affecting the overall level of - and ß-tubulin molecules.

To examine the effect of excess Alp31^A in a ß-tubulin mutant, pREP1-alp31⁺ was introduced into a ts nda3 strain. As shown in Fig 5C, overproduction of Alp31^A resulted in inhibition of colony formation in the absence of thiamine. Similar experiments were performed in ts 2-tubulin mutants, but no strong toxicity was observed (Table 3). These results indicate that the cellular level of Alp31^A has to be precisely regulated, that either a loss or excess of the protein results in defects in microtubule integrity, and that Alp31^A shows genetic interactions with ß-tubulin, but not -tubulin.

Alp31^A does not coprecipitate with Alp1^D, Alp11^B, or tubulins:
As shown previously (RADCLIFFE et al. 1999 ), Alp11^B binds -tubulin in the cell. We sought to find out whether Alp31^A forms a stable complex with ß-tubulin. To this end, the chromosomal alp31⁺ gene was tagged with GST at its C terminus (MATERIALS AND METHODS). Tagging did not interfere with Alp31 function, as a haploid strain containing Alp31-GST did not show any discernible abnormality in cell morphology (PR27; Table 2). Immunoblotting using anti-GST antibody showed that a tagged strain specifically contains an immunoreactive band of 50 kD that corresponded to the predicted size of Alp31-GST (25 + 25 kD; Fig 6A). Also, a doubly tagged strain containing Alp1-HA and Alp31-GST was constructed (PR28). Using these strains, immunoprecipitation was performed with anti-GST antibody. Neither Alp1^D and Alp11^B nor -/ß-tubulin co-immunoprecipitated (Fig 6B, lanes 4 and 6, note that some level of - and ß-tubulins precipitated nonspecifically by binding to beads). Furthermore, reciprocal immunoprecipitation experiments using anti-HA as a primary antibody showed that Alp1^D does not coprecipitate with Alp31^A or - or ß-tubulin (lanes 10 and 12). This shows a clear difference between the properties of Alp31^A and of Alp11^B, which tightly binds -tubulin under the same conditions (RADCLIFFE et al. 1999 ).

View larger version (35K): In this window In a new window Download PPT slide   Figure 6. Characterization of the Alp31 protein. (A) Identification of the alp31+ gene product by chromosomal tagging. The chromosomal alp31+ gene was tagged with GST at its C terminus. Cell extracts were prepared from the parental (untagged) strain (lane 1) or three independent tagged strains (PR27, lanes 2–4), and immunoblotting was performed with anti-GST antibody. Alp31-GST (50 kD) is emphasized with an arrow and a nonspecific band (22 kD) is marked with a black dot. The position of size markers is shown at the right. (B) Lack of interaction between Alp31 and the other cofactors and tubulins. Cell extracts were prepared from an untagged wild-type (lanes 1 and 7), singly tagged (Alp31-GST, PR27, lanes 2, 6, 8, and 12), or doubly tagged strain (Alp31-GST Alp1-HA, PR28, lanes 3, 4, 5, 9, 10, and 11). Immunoprecipitation was performed with either anti-GST (lanes 4, 6, and 7), anti-HA antibody (lanes 10 and 12), or mock treatment (beads only, lanes 5 and 11). Precipitated proteins were detected with anti-HA, anti-ß-tubulin, anti-GST, anti-Alp11 or anti--tubulin antibodies. Total cell extracts (corresponding to 1/30 amount used for immunoprecipitation) were also run (lanes 1, 2, 3, 8, and 9).

Alp31^A exists in a complex distinct from Alp1^D, Alp11^B, and tubulins:
Next, the native size of Alp31 was analyzed by gel filtration chromatography. As shown in Fig 7, it was found that Alp31^A did not cofractionate with any of the proteins examined (Alp1^D, Alp11^B, and tubulins) and existed predominantly in fractions between tubulins and Alp11^B (fractions 21–23; Fig 7). This is consistent with the previous results showing that Alp31^A forms a stable interaction with neither Alp11^B and Alp1^D nor tubulins (by immunoprecipitation; Fig 6B). It is of note that a subpopulation of Alp1^D, Alp11^B, and tubulins (and Alp21^E; RADCLIFFE et al. 1999 ) exists in a large complex (>20S), but this is not the case with Alp31^A, although at a smaller position (200–300 kD), Alp31^A appeared to cofractionate with -tubulin (lanes 21–23) and also ß-tubulin (RADCLIFFE and TODA 2000 ).

View larger version (24K): In this window In a new window Download PPT slide   Figure 7. Size fractionation of the Alp31 protein. Gel filtration chromatography. Soluble extracts from a doubly tagged strain (Alp31-GST Alp1-HA, PR28) were separated through a Superose-6 column and fractions were analyzed by immunoblotting with anti-Alp11, anti-GST, anti-HA, and anti--tubulin antibodies. Total extract (10 µg) was also run (far left lane). Numbers above (1–35) correspond to fraction numbers. Positions of size markers (43, 232, 669, and 2000 kD) are also shown.

Taken together, the data show that Alp31^A plays an important role in microtubule integrity; however, compared to Alp11^B, Alp21^E, and Alp1^D, which are indispensable for maintaining -tubulin levels, microtubule structures, and cell survival, its requirement is less stringent.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our analysis highlights a clear differentiation between - and ß-tubulins in their requirement for cofactors in the microtubule biogenesis pathways. We have shown that cells lacking Alp31^A, the cofactor A homolog (structural and functional), grow and divide despite morphological defects. The possibility that fission yeast has another homolog for cofactor A is not formally excluded; however, it is unlikely, as a homology search against the fission yeast genome database shows Alp31 is the only homolog (>98% of the genomic sequence has been completed; the Sanger Centre, Hixton, UK). Nonessentiality of Alp31^A is in clear contrast to findings for Alp11^B, which is absolutely required for cell division and viability (Fig 8). It has been proposed that cofactor A is a cochaperonin, which interacts with ß-tubulin released from chaperonin complexes (GAO et al. 1994 ). Furthermore by analogy to the role of cofactor B for -tubulin (TIAN et al. 1997 ; RADCLIFFE et al. 1999 ), cofactor A may function to maintain a reservoir of partially folded ß-tubulins (ARCHER et al. 1995 ; TIAN et al. 1996 ). This explains why overproduction of Alp31^A results in the disappearance of intact microtubule structures associated with cell polarity defects; excess Alp31^A may sequester a pool of free ß-tubulin molecules. Consistent with this, we have shown that the overproduction of Alp31^A results in greater toxicity in strains containing mutations in ß-tubulin. Compared to Alp11^B, which contains a conserved motif (CLIP-170 domain) responsible for binding -tubulin (LEWIS et al. 1997 ; FEIERBACH et al. 1999 ; RADCLIFFE et al. 1999 ), the amino acid sequence of Alp31^A does not give any significant clues as to putative protein-protein interactions, except that it contains two coiled-coil domains with structural similarities to the DnaJ protein (this study; LLOSA et al. 1996 ; MELKI et al. 1996 ). It seems likely that the binding properties of cofactor A/ß-tubulin and cofactor B/-tubulin differ at the molecular level.

View larger version (13K): In this window In a new window Download PPT slide   Figure 8. Essential and nonessential pathways for in vivo microtubule biogenesis. The two pathways required for microtubule biogenesis in fission yeast are shown. Small shaded circles surrounding - and ß-tubulin molecules represent the chaperonin complex. The linear sequential pathway (top) in which the cofactor homologs Alp11B, Alp21E, and Alp1D are involved mainly functions to control -tubulin availability. Alp41 is a conserved GTPase, which functions prior to Alp1D, probably in concert with Alp21E (RADCLIFFE et al. 2000 ). Without these proteins, cells fail to maintain normal -tubulin levels and lose microtubule structures, resulting in cell division defects. On the other hand, the role of Alp31A in microtubule integrity, however, is dispensable. Alp31 is most likely to function, like its homologs in other organisms, in the ß-tubulin-dependent pathway.

Genetic analysis highly suggests that Alp31^A regulates the ß-tubulin pathway, rather than the -tubulin pathway, in which Alp11^B, Alp21^E, and Alp1^D are involved. This notion is consistent with the proposed role of cofactor A in the correct folding of ß-tubulin (LEWIS et al. 1997 ). alp31-deleted cells have compromised microtubules; no intact cytoplasmic microtubules were observed under immunofluorescence microscopy. There is evidence that fission yeast cells can grow without cytoplasmic microtubules (SAWIN and NURSE 1998 ). However, mitotic spindles are absolutely essential for cell division. It is possible that Alp31^A is mainly involved in the integrity of cytoplasmic microtubules and its requirement for spindle formation is less stringent. The reason that Alp31^A is dispensable, therefore, might be ascribable to the differential requirement for Alp31^A between cytoplasmic and spindle microtubules. alp31 cells are sensitive to ectopic overexpression of Alp21^E, the basis of which is currently unknown. It is possible that further unbalance of the stoichiometry of molecules between functional - and ß-tubulins, caused by the combination of overproduced Alp21^E and the absence of Alp31^A, is deleterious for the cell.

The in vivo dispensability of Alp31^A may correlate to the previous results reported for the folding pathways in vertebrates from in vitro data. In this system, although cofactor A binds partially folded ß-tubulin intermediates, it does not participate in folding reactions per se when reactions are performed using purified components (TIAN et al. 1996 ). In contrast the requirement of cofactor B is more stringent both in vitro and in vivo (RADCLIFFE et al. 1999 ). It is possible that, upon release from chaperonin complexes, unlike -tubulin, ß-tubulin can be folded by itself to some extent without cofactor A, such that it is in a conformational state capable of interacting with other cofactors and -tubulin. This view is consistent with the fact that ß-tubulin appears more stable than -tubulin in the absence of cofactor function in fission yeast cells (this study; RADCLIFFE et al. 1999 ) and that overproduction of ß-tubulin is in general more toxic than that of -tubulin (HIRAOKA et al. 1984 ; BURKE et al. 1989 ; KATZ et al. 1990 ; WEINSTEIN and SOLOMON 1990 ).

We failed to see a physical interaction between Alp31^A and ß-tubulin by immunoprecipitation, as opposed to results reported for counterparts in other organisms (mammalian cofactor A and budding yeast Rbl2; ARCHER et al. 1995 , ARCHER et al. 1998 ; LEWIS et al. 1997 ; VEGA et al. 1998 ). Gel filtration chromatography shows that an interaction between Alp31^A and ß-tubulin might be either weaker or more delicate in fission yeast. Given the defective phenotypes of alp31-deleted cells, however, it is unambiguous that alp31⁺ is involved in microtubule integrity in the cell. These defects include altered cell shape (bent or branched), synthetic lethal interaction with mutations in ß-tubulin, hypersensitivity to the overproduction of, usually nontoxic, Alp21^E, and unstable microtubule structures. The cell shape defects seen in alp31 cells are reminiscent of microtubule defects in fission yeast (UMESONO et al. 1983B ; HIRAOKA et al. 1984 ; MATA and NURSE 1997 ; RADCLIFFE et al. 1998 ; SAWIN and NURSE 1998 ). It is of note that, although overproduction of Alp21^E has no discernible effects in wild-type cells (RADCLIFFE et al. 1999 ), it is toxic in the cold-sensitive nda2-52 mutant (defective in 1-tubulin; GRISHCHUK and MCINTOSH 1999 ). Therefore, although the cellular levels of both - and ß-tubulin molecules appear normal, the quality of tubulins must be somehow compromised in the absence of Alp31^A.

In summary we have shown that the cofactor-dependent microtubule biogenesis in vivo requires two separate pathways, one essential (Alp11^B-Alp21^E-Alp1^D) and the other nonessential (Alp31^A; Fig 8). These results strongly imply that - and ß-tubulins should not be regarded simply as evolutionarily duplicated partners, instead these two proteins must be functionally and biochemically distinct. It is well established that, in addition to their asymmetrical positioning in /ß-heterodimers along microtubule polymers, with regards to GTP binding, these two proteins differ; while -tubulin-GTP is nonexchangeable, GTP bound by ß-tubulin is exchangeable and also hydrolyzable (BURNS and FARRELL 1996 ). These differences may explain why most of the antimitotic drugs acting through microtubules, such as thiabendazole compounds, colchicine, and paclitaxel, target ß-tubulin, rather than -tubulin (UMESONO et al. 1983B ; RAO et al. 1994 ; BAI et al. 1996 ). The screening of new drugs that affect microtubule integrity via cofactor functions might be a novel avenue for agriculture or pharmaceutics toward the improvement of food production or remedies for microtubule-related human diseases.

	FOOTNOTES

¹ Present address: Oxford Biomedica (UK) Ltd., Oxford OX4 4GA, United Kingdom.

	ACKNOWLEDGMENTS

We thank Drs. Kate Compton, Keith Gull, Paul Nurse, Frank Solomon, and Hiroyuki Yamano for providing materials used in this study. We thank Dr. Jacqueline Hayles for critical reading of the manuscript and useful suggestions. M.A.G. was supported by a European Molecular Biology Organization long-term fellowship.

Manuscript received December 18, 1999; Accepted for publication May 22, 2000.

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