Structure–Function Relationships in Yeast Tubulins

Abstract

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.

INTRODUCTION

Microtubules are ubiquitous cytoskeletal structures, made up of heterodimers of α and β-tubulin (Hyams and Lloyd, 1994). Microtubules have been studied in many eukaryotes, both in vitro and in vivo, with the aim of defining both the functions of microtubules and the spatial and temporal regulation of those microtubule functions. As in all eukaryotic cells, microtubules are necessary for chromosome movement in Saccharomyces cerevisiae. Unlike most other eukaryotes, the only other functions known to depend on microtubules in yeast are the movement of nuclei to the bud neck before mitosis and the fusion of nuclei after mating (Huffaker et al., 1988; Jacobs et al., 1988). The high degree of conservation among tubulin proteins (Little and Seehaus, 1988; Burns, 1991), together with the relative simplicity of the yeast microtubule cytoskeleton, make S. cerevisiae a suitable organism for studying the microtubule cytoskeleton.

TUB1 is the major gene encoding α-tubulin in S. cerevisiae (Schatz et al., 1986a,b), and TUB2 is the only gene encoding the β-tubulin gene (Neff et al., 1983). TUB3 is a second gene encoding α-tubulin; it is expressed at lower levels than TUB1 (Schatz et al., 1986a,b). TUB1 and TUB2 are essential genes_, whereas a strain carrying a deletion of TUB3 is viable. tub3 null mutations, and most tub1 mutations, show a characteristic supersensitivity to the benzimidazole microtubule drug benomyl (Neff et al., 1983; Schatz et al., 1986b). Deletion of TUB1 produces a dominant phenotype resulting from haploinsufficiency; heterozygotes tend to become trisomic for the wild-type chromosome XIII, which presumably provides a growth advantage because of an additional copy of both TUB1 and TUB3 (Schatz et al., 1986b). The two genes encoding α-tubulin in yeast are very similar, producing protein products that are ∼90% identical, making them much more similar to each other than to α-tubulin proteins from other organisms, to which they are ∼70% identical (Little and Seehaus, 1988). Either α-tubulin gene, TUB1 or TUB3, can compensate for loss of the other, if expressed at high enough levels. Therefore, the differences between TUB1 and TUB3 appear to be largely quantitative, resulting from higher levels of TUB1 expression, rather than qualitative (i.e., sequence) differences in the proteins (Schatz et al., 1986b).

Clustered charged-to-alanine scanning mutagenesis has been used to mutagenize the surface of proteins systematically (Bass et al., 1991; Bennett et al., 1991; Gibbs and Zoller, 1991). This strategy has been used previously to mutagenize the two other major cytoskeletal proteins in S. cerevisiae, actin and β-tubulin (Wertman et al., 1992; Reijo et al., 1994). In the case of ACT1 (the single yeast gene encoding actin), such a program of mutagenesis produced many conditionally lethal alleles, which had previously proven quite difficult to isolate (Wertman et al., 1992). In the case of TUB2 (the sole β-tubulin gene), many new conditional alleles were obtained, some displaying previously unreported phenotypes (Reijo et al., 1994). Both of these sets of mutants are proving to be useful in studies of the cytoskeleton in yeast (Pasqualone and Huffaker, 1994; Amberg et al., 1995; Cali et al., 1998).

Recently the structure of bovine tubulin has been solved at atomic resolution (Nogales et al., 1998b). This allows the results of years of study of the tubulin genes and the function of microtubules in yeast to be analyzed in light of the three-dimensional structure of the tubulin protein. In this paper we map nearly all the known systematic mutations in TUB1 and TUB2 to a model of yeast tubulin based on the bovine structure. As had been found with actin and cofilin (Wertman et al., 1992; Lappalainen et al., 1997), this mapping has indicated regions of the tubulin surface implicated in particular phenotypes, namely cold sensitivity and benomyl resistance. In addition, we have found evidence for the involvement of particular residues in binding of the microtubule ligand Bim1p (Schwartz et al., 1997), and others have found similar evidence for other proteins that bind tubulins (Feierbach et al., 1999).

MATERIALS AND METHODS

Media and Genetic Manipulations

Yeast media and techniques are as described (Rose et al., 1990), except that YPD medium was supplemented with adenine sulfate to a concentration of 40 mg/l. Minimal medium was supplemented only with the required amino acids, rather than using synthetic complete medium, unless specified. Permissive temperature for all experiments was 25°C. Sporulation was induced by diluting a liquid culture 100-fold into sporulation medium described by Kassir and Simchen (1991). Benomyl, a generous gift from DuPont (Wilmington, DE), was kept as a 10 mg/ml stock in dimethylsulfoxide at −20°C. It was added to warm medium, with vigorous swirling to prevent precipitation of the benomyl, just before pouring plates. Bacterial media and techniques were as described (Sambrook et al., 1989). Ampicillin was used at a final concentration of 50 μg/ml.

Strains and Plasmids

Plasmids are listed in Table 1, along with details of their construction. Yeast strains were derived from YPH102 and YPH250 (Sikorski and Hieter, 1989) and are listed in Table 2. YPH102 was transformed with a TUB1 CEN plasmid, pRB326. The resulting strain, KRY73, was transformed with a tub1 deletion construct from pRB2067, to give DBY6590. DBY6591 was constructed by transforming YPH250 with the TUB1-LYS2 construct from pRB2070. DBY6590 was crossed to DBY6591 to give the diploid KRY76. KRY76 was dissected to remove the trp1-Δ1 allele, and two resulting spores, DBY6592 and DBY6593, were crossed, yielding DBY6596. DBY6596 was the recipient strain for the Ala-scan alleles, and its genomic configuration was verified by Southern blot analysis. Mating type tester strains DBY786 and DBY789 were essentially congenic with S288C. Bacterial strains used for transformation and propagation of plasmid DNA were either DH5α or DH5αF′ (Sambrook et al., 1989).

Table 1.

Plasmids used in this study

Plasmid	Description	Source or reference
pUC119	pUC19 with M13 origin of replication	Sambrook et al., 1989
pRB306	TUB1 in pRB322	Schatz et al., 1986b
pRB326	TUB1 in YCp50	Schatz et al., 1986b
pJJ217	HIS3 in pUC18 polylinker	Jones and Prakash, 1990
pJJ282	LEU2 in pUC18 polylinker	Jones and Prakash, 1990
pRS317	LYS2 in pRSS56	Sikorski and Hieter, 1989
pRB2063	TUB1 fragment (SphI–SacI) from pRB306 inserted into SphI–SacI-cut polylinker of pUC119	This study
pRB2065	BamHI–SalI fragment from pJJ282 (LEU2 gene) Klenow filled and inserted into the HpaI site of pRB2063, downstream of TUB1; LEU2 is oriented in the same direction as TUB1	This study
pRB2067	SalI–KpnI (partial) fragment from pJJ217 containing the entire HIS3 gene and flanking regions, inserted into XhoI–KpnI vector fragment from pRB2063; HIS3 is in the same orientation that TUB1 was; note that this disruption construct removes the first 763 bp of the 1580-bp ORF downstream of TUB1 (YML086c)	This study
pRB2070	PvuII fragment from pRS317 containing LYS2 gene inserted into HpaI site of pRB2063; LYS2 is oriented in the same direction as TUB1	This study
pRB2666	BglII fragment containing TUB3 from pRB300 ligated into BamHI site of pUC119	This study
pRB2668	SalI–SmaI containing HIS3 fragment from pJJ217 ligated into XhoI–NdeI (Klenow-filled) vector fragment from pRB2666; HIS3 is in the same orientation as TUB3 was	This study
pRB2254 through pRB2412	pRB2065 mutagenized via site-directed mutagenesis, yielding three isolates of each Ala-scan mutation, tub1-801 through tub1-853	This study

Table 2.

Yeast strains used in this study

Strain	Genotype	Source or reference
YPH102	MAT∝ ade2-101 his3-Δ200 lys2-801 leu2-Δ1 ura3-52	Sikorski and Hieter, 1989
YPH250	MATa ade2-101 his3-Δ200 lys2-801 leu2-Δ1 ura3-52 trp1-Δ1	Sikorski and Hieter, 1989
DBY786	MAT∝ trp1-1	This laboratory
DBY789	MATa trp1-1	This laboratory
KRY73	MAT∝ ade2-101 his3-Δ200 lys2-801 leu2-Δ1 ura3-52	This study
	plasmid: pRB326
DBY6590	MAT∝ ade2-101 his3-Δ200 lys2-801 leu2-Δ1 ura3-52 tub1Δ::HIS3	This study
	plasmid: pRB326
DBY6591	MATa ade2-101 his3-Δ200 lys2-801 leu2-Δ1 ura3-52 trp1-Δ1 TUB1-LYS2	This study
KRY76 (DBY6590 × 6591)	MATa/∝ ade2-101/ade2-101 his3-Δ200/his3-Δ200 lys2-801/lys2-801 leu2-Δ1/leu2-Δ1 ura3-52/ura3-52 trp1-Δ1/TRP1 TUB1-LYS2/tub1Δ::HIS3	This study
	plasmid: pRB326
DBY6592	MAT∝ lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1-LYS2	This study
	plasmid: pRB326
DBY6593	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1Δ::HIS3	This study
	plasmid: pRB326
DBY6596	MATa/∝ lys2-801/lys2-801 ade2-101/ade2-101 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1 ura3-52/ura3-52 TUB1-LYS2/tub1Δ::HIS3	This study
	plasmid: pRB326
DBY6598	MAT∝ lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1-LYS2 tub3Δ::HIS3	This study
	plasmid: pRB326
DBY6599	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1-LYS2 tub3Δ::HIS3	This study
	plasmid: pRB326
DBY6600	MATa/∝ lys2-801/lys2-801 ade2-101/ade2-101 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1 ura3-52/ura3-52 TUB1-LYS2/TUB1-LEU2	This study
	plasmid pRB326
DBY6601–6653	Like DBY6600, but with tub1 Ala-scan alleles, -801 to -853, respectively	This study
DBY6654	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1–LEU2	This study
DBY6655	MAT∝ lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1–LEU2	This study
DBY6656–6761	Like DBY6654 and 6655, but with tub1 Ala-scan alleles, -801 to -853, respectively (2 each, 1 of each mating type)	This study
DBY6762	MATa/∝ lys2-801/lys2-801 ade2-101/ade2-101 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1 ura3-52/ura3-52 TUB1-LYS2/TUB1-LEU2 tub3Δ::HIS3/TUB3	This study
	plasmid: pRB326
DBY6816	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1–LEU2 tub3Δ::HIS3	This study
DBY6822	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-803–LEU2 tub3Δ::HIS3	This study
DBY6824	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-804–LEU2 tub3Δ::HIS3	This study
DBY6826	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-805–LEU2 tub3Δ::HIS3	This study
DBY6828	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-806–LEU2 tub3Δ::HIS3	This study
DBY6830	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-807–LEU2 tub3Δ::HIS3	This study
DBY6838	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-811–LEU2 tub3Δ::HIS3	This study
DBY6840	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-812–LEU2 tub3Δ::HIS3	This study
DBY6846	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-815–LEU2 tub3Δ::HIS3	This study
DBY6854	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-819–LEU2 tub3Δ::HIS3	This study
DBY6866	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-825–LEU2 tub3Δ::HIS3	This study
DBY6868	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-826–LEU2 tub3Δ::HIS3	This study
DBY6878	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-831–LEU2 tub3Δ::HIS3	This study
DBY6880	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-832–LEU2 tub3Δ::HIS3	This study
DBY6882	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-833–LEU2 tub3Δ::HIS3	This study
DBY6886	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-835–LEU2 tub3Δ::HIS3	This study
DBY6894	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-839–LEU2 tub3Δ::HIS3	This study
DBY6898	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-841–LEU2 tub3Δ::HIS3	This study
DBY6902	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-843–LEU2 tub3Δ::HIS3	This study
DBY6916	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-850–LEU2 tub3Δ::HIS3	This study
DBY6918	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-851–LEU2 tub3Δ::HIS3	This study
DBY6920	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 tub1-852–LEU2 tub3Δ::HIS3	This study
DBY7830	MAT∝ lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1-LYS2 tub2-201	Schwartz et al., 1997
	plasmid: pRB326
DBY7051	MATa ACT1::HIS his3-Δ200 leu2-3,112 ura3-52 ade4 tub2-201	This laboratory
DBY8154	MATa lys2-801 ade2-101 his3-Δ200 leu2-Δ1 ura3-52 TUB1-LEU2 tub2-201	This study

To construct the tub3Δ strain, DBY6592 was transformed with the gel-purified 2.8-kb BstEII–HindIII fragment from pRB2668, obtained from a BstEII digest followed by a partial HindIII digest, to give DBY6598. The proper genomic organization of the strain was verified by Southern blot analysis. To obtain a congenic strain of the opposite mating type, DBY6598 was mated to DBY6654, yielding the haploid His⁺ Lys⁺ Ura⁺ segregant DBY6599.

Two haploids with each tub1 mutant, one of each mating type, were mated to a tub3Δ strain; MATa tub1 strains were mated to DBY6598, and MATα tub1 strains were mated to DBY6599. The tub1 tub3Δ double mutants (DBY6816 through DBY6920) are haploid progeny of these crosses.

DNA Manipulation

All restriction enzymes, T4 ligase, T7 polymerase, and Klenow were from New England Biolabs (Beverly, MA). Calf intestinal phosphatase was from Boehringer Mannheim (Indianapolis, IN). Taq polymerase was from Perkin Elmer-Cetus (Foster City, CA) or Life Technologies (Santa Clara, CA). Buffers were either used as supplied or remade according to the manufacturer's specifications. Nucleotides were from Pharmacia (Alameda, CA). Oligonucleotide primers were supplied by Genset (La Jolla, CA).

Site-directed Mutagenesis

Methods were modified from those of Kunkel et al. (1987) and Sambrook et al. (1989) and are described in detail elsewhere (Miller et al., 1996). Briefly, an oligonucleotide bearing the desired mutation (see Table 3) was annealed to a single-stranded DNA template, pRB2065, containing a high frequency of uracil misincorporations. Oligonucleotides were designed with at least 12 bp of homology on either side of the altered nucleotides. Synthesis of the second strand, primed by the mutant oligonucleotide, was done in vitro, and the product was transformed into an Escherichia coli strain able to recognize and repair the uracil misincorporations. The repair process resulted in coding information from the template (wild-type) strand being replaced with the coding information from the mutant strand. Resultant plasmids were screened for the introduction or loss of a restriction site to identify those with the desired mutation.

Table 3.

Summary of mutations

tub1 allele	Amino acid substitutions	DNA sequence changesa	Diagnostic restriction sites	Phenotypeb
801	R2A, E3A	GCTGCA	Gain PstI	Cs, Ts, BenSS
		AGAGAA
802	E27A, H28A	GCAGCC	Gain BbvI	RL
		GAGCAC
803	K31A, D33A	GCGCCGGCT	Gain HinP1I,	BenSS
		AAGCCGGAT	Lose BsaBI
804	H35A, E37A, D38A	GCTCTAGCAGCT	Gain PvuII,	BenSS
		CATCTAGAAGAT	Lose BsaBI
805	K42A, K44A	GCGCCGGCG	Gain HinP1I	BenSS
		AAGCCGAAG
806	E47A, E48A	GCAGCT	Gain PvuII	BenR
		GAAGAG
807	H55A, E56A	GCTGCA	Gain BbvI	BenSS
		CATGAA
808	K61A, R65A	GCGTTCGTTCCAGCT	Gain PvuII	RL
		AAGTTCGTTCCAAGG
809	D70A, E72A	GCTTTAGCG	Gain HinP1I	Slow growth,
		GATTTAGAG		BenSS
810	D77A, E78A, R80A	GCAGCAGTCGCT	Gain BbvI	BenSS
		GACGAAGTCCGT
811	K85A, D86A	GCTGCC	Gain BbvI	WT
		AAGGAC
812	H89A, E91A	GCTCCAGCA	Gain BpmI	BenSS
		CATCCAGAA
813	K97A, E98A, D99A	GCGGCGGCC	Gain NotI	Slow growth,
		AAGGAGGAC		BenSS
814	R106A, H108A	GCTGGCGCT	Gain PvuII	Slow growth,
		AGAGGCCAT		BenSS
815	R113A, E114A	GCAGCA	Gain BbvI	WT
		AGAGAA
816	D118A, D121A, R122A	GCTGTTCTGGCTGCG	Gain BbvI	Cs, BenSS
		GATGTTCTGGATAGG
817	R124A, K125A	GCAGCA	Gain BbvI	BenSS
		AGAAAA
818	D128A, D131A	GCCCAATGTGCA	Gain BsgI	Cs, BenSS
		GACCAATGTGGAT
819	T146A	GCA	Gain BsgI,	BenSS
		ACT	BspMI
820	E156A, E157A	GCAGCA	Gain BbvI	DL
		GAAGAA
821	E161A, K164A, K165A	GCATACGGTGCGGCA	Gain Fnu4HI	BenSS
		GAATACGGTAAGAAA
822	K167A, E169A	GCGCTGGCA	Gain HinP1I	Slow growth,
		AAGCTGGAA		BenSS
823	E197A, H198A, D200A	GCAGCTGCAGCT	Gain PstI, PvuII	RL
		GAACATGCAGAT
824	D206A, E208A	GCGAATGCG	Gain NruI,	Cs, BenSS
		GATAATGAG	BsmI
825	D212A, K215A, R216A	GCCATGTGCGCAGCA	Gain BbvI	BenR
		GACATGTGCAAAAGA
826	D219A, R222A	GCTATCCCAGCA	Lose EcoRV	BenR
		GATATCCCAAGA
827	R244A, D246A	GCATTCGCG	Gain BstUI	Slow growth,
		AGATTCGAG		BenSS
828	D252A, E255A	GCTTTGAACGCG	Gain MluI	DL
		GATTTGAACGAA
829	R265A, H267A	GCAATTGCT	Gain MunI,	Slow growth,
		AGAATTCAT	Lose EcoRI	BenSS
830	K279A, K281A	GCATCAGCT	Gain PvuII	BenSS
		AAATCAAAG
831	H284A, E285A	GCTGCG	Gain Fnu4HI,	Cs, BenSS
		CATGAG	Lose HinfI, PleI
832	K305A, D307A	GCGTGTGCA	Gain ApaLI	BenR
		AAGTGTGAT
833	R309A, D310A, K312A	GCAGCTGGTGCA	Gain PvuII,	BenSS
		AGAGATGGTAAA	Fnu4HI
834	R321A, D323A	GCGGGTGCT	Gain BstUI	Slow growth,
		AGGGGTGAT		BenSS
835	R327A, D328A,	GCTGTTCAAGCA	Gain PvuII	WT
	R331A	GATGTTCAAAGA
836	E334A, K337A	GCGCAGGTGGCA	Gain BstUI,	Cs, Ts, BenSS
		GAGCAGGTGAAA	HinP1I
837	K339A, K340A	GCGGCG	Gain Fnu4HI,	BenSS
		AAGAAG	BstUI, Lose  BbsI
838	D373A, R374A	GCTGCA	Gain PstI, SfcI	BenSS
		GATAGG
839	E387A, K390A, R391A	GCAGCTTGGGCGGCA	Gain PstI	BenR
		GAGGCTTGGAAGAGA
840	D393A, R394A, K395A, D397A	GCTGCAGCATTCGCT	Gain PstI,	RL
		GATAGAAAATTCGAT	BsmI, SfcI,  Lose ClaI
841	D393A, R394A	GCTGCA	Gain PstI	BenR
		GATAGA
842	K395A, D397A	GCATTCGCT	Gain BsmI	Cs, Ts, BenSS
		AAATTCGAT
843	K395A	GCA	Gain BsmI	WT
		AAA
844	D397A	GCA	Gain AseI	BenSS
		GAT
845	K402A, R403A	GCAGCT	Gain PvuII	Slow growth, BenSS
		AAACGT		BenSS
846	E412A, E415A	GCAGGTATGGCA	Gain BsgI,	Cs, Ts, BenSS
		GAAGGTATGGAA	BspMI
847	E416A, E418A	GCAGGTGCA	Gain BsmI,	Slow growth,
		GAAGGTGAA	BspMI, Lose  EcoRI	BenSS
848	E421A, R423A,	GCAGCTGCAGCAGCT	Gain PstI, PvuII	Slow growth,
	E424A, D425A	GAAGCTAGAGAAGAT		BenSS
849	E430A, R431A, D432A	GCTGCAGCT	Gain PstI	Slow growth,
		GAAAGAGAT		BenSS
850	E435A, D439A	GCGGTGGGTGCCGCC	Gain BstUI	BenSS
		GAAGTGGGTGCCGAC
851	E443A, E444A, E445A,	GCTGCAGCGGCA	Gain PstI	BenSS
	E446A	GAGGAAGAGGAA
852	F447A	GCT	Gain HindIII	BenSS
		TTT
853	E184A	GCG	Gain HinP1I	Slow growth,
		GAG		BenSS

^aThe mutant sequence is above the corresponding wild-type sequence. Altered nucleotides are in bold; altered codons are underlined. 

^bBenR, benomyl resistant; BenSS, benomyl supersensitive; Cs, cold-sensitive; DL, dominant lethal; RL, recessive lethal; Slow growth, impaired growth at all temperatures; Ts, heat-sensitive; WT, wild-type. 

Construction of tub1 Strains

tub1 mutant plasmids, digested with SphI and SacI to release the fragment containing the tub1 and downstream LEU2 integration, were transformed into DBY6596, and Leu⁺ transformants were selected. DBY6596 is a heterozygous diploid with a HIS3-marked tub1 deletion (tub1Δ::HIS3) on one chromosome XIII; on the homologue, the wild-type TUB1 locus marked by LYS2 integrated downstream. In this diploid, a TUB1 centromeric plasmid, pRB326, is maintained to prevent the aneuploidy that results from propagating strains heterozygous for a tub1 deletion (Schatz et al., 1986b). The transforming tub1-LEU2 fragment could integrate by homologous recombination at any of the three TUB1 loci (two chromosomal and one plasmid) in the recipient strain. The desired integration event was recovered by screening Leu⁺ transformants that had become His⁻ as a result of the replacement of the tub1Δ::HIS3 allele. This screening also eliminated any unwanted products of illegitimate recombination (i.e., integrations at loci other than TUB1).

The resultant diploids are heterozygous for the tub1 mutation. In addition, both the wild-type and mutant TUB1 loci are marked by LYS2 and LEU2, respectively. These auxotrophic markers were inserted ∼1 kb downstream of TUB1 and therefore remain tightly linked to TUB1 during meiotic segregation, allowing for convenient identification of tub1-containing haploid segregants. These markers can also readily reveal any chromosome XIII aneuploidy, because Lys⁺ and Leu⁺ do not segregate 2:2 under those conditions. It should be noted that insertion of these auxotrophic marker genes disrupts the open reading frame downstream of TUB1. This gene, ALO1, encodes d-arabinono-1,4-lactone oxidase, which catalyzes the biosynthesis of d-erythroascorbic acid in yeast (Huh et al., 1998). This disruption had no microtubule phenotype on its own, and all tub1 alleles were compared with the appropriate control, a strain with a wild-type TUB1 gene marked by the downstream auxotrophic marker insertion in ALO1.

To assess viability in the absence of the plasmid-borne TUB1 gene, the heterozygous diploids and the dissection plates containing haploid segregants were replica plated to medium containing 5-fluoroorotic acid (5-FOA), which selects against the URA3-marked pRB326 plasmid. Each dissection plate was also replica plated to mating-type tester lawns and onto media to test for the various auxotrophic markers. The auxotrophic markers indicate which haploid segregants carry pRB326 and whether they carry the wild-type (Lys⁺) or mutant (Leu⁺) TUB1 gene.

Each mutant was constructed twice independently. Both independent isolates of each haploid mutant, and occasionally the heterozygous diploid parent, were verified by PCR amplification of the TUB1 gene followed by a restriction digest to verify the presence of the restriction site introduced by that particular mutation (Table 3). This test also confirmed the absence of the wild-type TUB1 gene in the haploids.

Yeast Transformation

Yeast were transformed using electroporation (Becker and Guarente, 1991), using ∼1–2 μg of DNA per transformation. For constructing parental strains and for making the first isolate of each Ala-scan mutant, the DNA usually came from a gel-purified fragment. The DNA was purified using the Qiaex (Qiagen, Chatsworth, CA) DNA purification system. For the second isolate of each mutation (and sometimes for the first isolate as well), the DNA was digested from a crude minipreparation of DNA, ethanol precipitated, and transformed directly without purification of the transforming fragment.

Colony PCR to Verify Mutation

Genomic DNA was amplified directly from yeast colonies by resuspending a colony directly into 5 μl of water in a PCR tube and immediately placing it on ice. A mixture of all the other components was prepared on ice. The mixture was composed such that adding 25 μl to the 5 μl of cells would produce a final concentration of 0.25 μM of each primer, 2.5 U of Taq polymerase, 1× Taq buffer (10 mM Tris-HCl, pH 8.5, 50 mM KCl, and 1.5 mM MgCl₂), and 0.1 μg/μl BSA. Immediately after dividing the mixture into each reaction tube while on ice, the tubes were vortexed very briefly, mineral oil was added, and tubes were placed into a preheated (>90°C) DNA Thermal Cycler (Perkin Elmer-Cetus). The cycling conditions varied but worked best with 94°C, 4 min, 30 cycles (92°C, 1 min; 55°C, 1 min; and 72°C, 2 min); 72°C, 20 min; and a 4°C soak.

Half of the reaction product (15 μl) was digested with the appropriate restriction enzyme (see Table 3) in a final reaction volume of 20 μl, and the entire reaction was run on an agarose gel to verify the presence or absence of the restriction site resulting from the particular tub1 mutation.

DNA Sequencing of tub2-201

tub2-201 DNA was PCR amplified from strains DBY7051 (twice) and DBY8154 (once) using Taq polymerase (Life Technologies, Rockville, MD) and primers KStub2-01 (5′-GCGCCATGGAGATGAGAGAAATCATTCAT-3′) and KStub2-02 (5′-GCGCCATGGTTATTCAAAATTCTCAGTGATTGG-3′), which anneal to the first and last 18 nucleotides of the TUB2 open reading frame. Products from independent amplifications were cloned into the NcoI site of pACTII (Bai and Elledge, 1996), resulting in three tub2-201-containing plasmids. The 5′- and 3′-most ends of the tub2-201 open reading frame were PCR amplified using primers KStub2-03 (5′-GCGGGATCCGTAGTGGTGAGGCAATTGG-3′), KStub2-04 (5′-GCGTCTAGACGAGATCAAAAGC-GTACC-3′), KStub2-05 (5′-GCGGGATCCCCCCAACAATGTGCA-AAC-3′), and KStub2-06 (5′-GCGTCTAGAGTTTATTTTGCTCCA-AGTGC-3′). Purified tub2-201 DNA (plasmid or PCR product) was sequenced with ThermoSequenase dye terminator cycle sequencing mix (Amersham, Arlington Heights, IL) and an ABI PRISM 373XL DNA sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

Immunofluorescence Microscopy

Visualization of microtubules with indirect immunofluorescence and 4′,6′-diamidino-2-phenylindole (DAPI) staining to visualize nuclei were performed as described previously (Schwartz et al., 1997).

Growth Rate Measurement

Growth rates were determined by diluting 1 ml of a dense YPD culture into 24 ml of YPD, incubating with agitation for 2–3 h to allow cells to begin growing exponentially, and then splitting the culture three ways: 8 ml of culture into 17 ml of YPD to be incubated at 11, 25, and 37°C. Readings were taken periodically with a Klett (New York, NY) colorimeter, and growth was plotted using Cricket Graph 1.3.2 (Cricket Software, Malvern, PA). The equation of the growth curve was used to calculate a doubling time for each strain.

Bud Index Determination

Cells were diluted from a saturated culture and incubated 3–4 h to allow cells to begin exponential growth. The cultures were then split and placed at 25 and 11°C. After two wild-type doubling times, the cells were counted using a hemacytometer. The bud index was simultaneously obtained by counting at least 200 cells and categorizing them as unbudded, small-budded, medium-budded (a bud diameter greater than one-fourth of the diameter of the mother cell body), or large-budded (a bud diameter greater than one-half of the diameter of the mother cell body).

Spot Replica Plating

Cells were grown in 2 ml of YPD for 36–48 h. to allow all cultures to arrive at approximately the same density. All cultures were diluted into wells of a microtiter dish, producing 10⁻¹ and 10⁻³ dilutions of each culture. These dilutions were plated using a 12-channel multipipettor, dispensing 3 μl per spot. Plates were observed and photographed daily to record data.

Molecular Modeling

The structure of the α–β-tubulin heterodimer from bovine brain was obtained by electron crystallography of zinc-induced tubulin sheets stabilized with taxol (Nogales et al., 1998b). Tub1p and Tub2p sequences were modeled with the LOOK software package (Molecular Applications Group, Palo Alto, CA) using the coordinates from the solved bovine tubulin structure as a template. Five hundred rounds of energy minimization refinements were used. GTP and GDP, but not taxol, were included in the model. The C termini of Tub1p (residues 442–447) and Tub2p (residues 428–457) were not included in the model, because they are not resolved in the current bovine tubulin structure (Nogales et al., 1998b). The resulting yeast tubulin model was quite similar to the bovine tubulin crystal structure: the root mean square deviation for 2601 polypeptide backbone atoms was 1.21 Å as calculated with Swiss-PdbViewer (Guex and Peitsch, 1997). The coordinates of this yeast tubulin model, along with interactive views of the model, are available at the Botstein laboratory web site: http://genome-www.stanford.edu/group/botlab/tubulin.

RESULTS AND DISCUSSION

Design and Construction of Charged-to-Ala Scan Mutations

Mutations were designed by examining the protein sequence of Tub1p, inspecting each window of five amino acids for two or more “charged” residues (we included H as well as K, R, D, and E). Mutations were designed to replace all of these “clustered charged” residues with alanine while minimizing the total number of mutations. As a result, each mutation changed two to four residues from charged to alanine. Only 10 charged residues were not included within any of the alanine-scanning mutations, and only one of these (H407) was within five amino acids of another charged residue. Residue E184, although not within a charged cluster, was mutated because it is in the middle of an extremely well-conserved sequence present in all tubulins (α, β, and γ): VVEPYN. tub1-842 was subdivided into two additional mutations, changing K395 and D397 separately (tub1-843 and tub1-844). Based on sequence conservation, these two residues may be important for α-tubulin-specific functions (Cleveland et al., 1990; Burns, 1991), and in addition, K395 has been shown to be an important residue for microtubule assembly (Sherman et al., 1983; Szasz et al., 1986).

Other noncharged residues were also mutated to alanine. T146 (mutated to A in tub1-819) is part of the tubulin signature sequence (GGGTGSG) (Bairoch, 1991), an invariant sequence found in every tubulin gene discovered to date. This sequence is now known to form part of the GTP-binding site by interacting with the nucleotide's phosphates (Nogales et al., 1998a,b). tub1-852 changes the last residue of Tub1p, a phenylalanine, to an alanine. This C-terminal residue is the subject of post-translational modification in vertebrate α-tubulins (Arce et al., 1978; Argarana et al., 1978; Barra et al., 1988).

During the construction of these mutations, two sequencing errors were discovered in the published TUB1 sequence (Schatz et al., 1986a). The first changes Leu-94(CTC) to Ile-94(ATC), and the second changes Thr-326 Gly-327(ACG GGA) to Thr-326 Arg-327(ACA AGA). Both of these amino acid changes increase the identity of Tub1p with Tub3p. Both of these nucleotide changes were also found by the systematic sequencing effort, as reported in the chromosome XIII sequence (Bowman et al., 1997).

A total of 53 mutations were constructed by site-directed mutagenesis and then integrated into the TUB1 locus by homologous recombination, resulting in tub1/TUB1 heterozygotes (see MATERIALS AND METHODS). Each mutant was constructed twice independently and was subjected to the same initial phenotypic characterization to be sure the phenotypes of the two isolates agreed. This guarded against misinterpretation attributable to second-site mutations introduced sometime during the mutagenesis procedure, because the likelihood of this occurring in two independent isolates is minimal. A TUB1 centromeric plasmid (pRB326) was maintained in these strains during their construction to prevent aneuploidy, which results from propagating strains heterozygous for tub1 null alleles (Schatz et al., 1986b). Once the mutants were constructed, the phenotypes of these diploids and their haploid progeny were assessed in the absence of the plasmid-borne TUB1 by plating on 5-FOA, which selects against the URA3-marked pRB326 plasmid.

tub1 Mutant Phenotypes

The phenotype of the tub1 haploids was characterized by testing for growth at different temperatures ranging from 11 to 37°C, and on media containing benomyl concentrations ranging from 2 to 50 μg/ml. Benomyl is a member of the benzimidazole family of microtubule-destabilizing drugs and is known to affect the growth of many tubulin mutants in yeast (Thomas et al., 1985; Schatz et al., 1988), although its exact mechanism of action is unknown. Growth rates in liquid medium were determined for each viable mutant at 11, 25, and 37°C. Each mutant was then classified as cold-sensitive (Cs) if growth was impaired at 11°C, temperature-sensitive (Ts) if growth was impaired at 37°C, and slow-growing if growth was impaired at 25°C as well as at extreme temperatures. In addition, each mutant was classified as benomyl-supersensitive if growth was impaired relative to wild type on media containing benomyl and benomyl-resistant if growth was better than wild type on media containing benomyl.

The phenotypes resulting from each tub1 mutation are shown in Figure 1 and Table 3. Two were dominant lethal mutations, because the diploid transformants, heterozygous for the mutation, were unable to grow without additional copies of TUB1 (pRB326) and therefore died on 5-FOA. Four were recessive lethal mutations; the haploid tub1 segregants failed to grow without the TUB1 plasmid on 5-FOA. Twenty-one alleles produced a conditional growth phenotype (Cs, both Cs and Ts, or impaired over the entire temperature range). Growth of each of these mutants at various temperatures is shown in Figure 2. Almost all of the mutations show an altered sensitivity to benomyl. Wild-type haploids (e.g., DBY6654 and DBY6655) grew well on concentrations of benomyl up to 20 μg/ml and grew weakly on a concentration of 30 μg/ml. Six strains, those containing the alleles tub1-806, -825, -826, -832, -839, and -841, grew well on 30 μg/ml benomyl and were therefore benomyl resistant. Despite previous extensive mutagenesis of TUB1 (Schatz et al., 1988), these are the first benomyl-resistant tub1 alleles isolated. These strains were not completely insensitive to benomyl, however, because they failed to grow on 75 μg/ml benomyl. Most of the other tub1 mutants were more benomyl sensitive than wild type, failing to grow on 20 μg/ml benomyl. We placed the tub1 mutants into five categories based on their benomyl sensitivities relative to wild type and the benomyl-supersensitive mutant tub3Δ (which failed to grow on 10 μg/ml benomyl). The categories were, in descending degree of sensitivity: 1) more sensitive than tub3Δ, 2) approximately equal in sensitivity to tub3Δ, 3) less sensitive than tub3Δ but more sensitive than wild type, 4) approximately equal in sensitivity to wild type, and 5) less sensitive than wild type (i.e., benomyl-resistant) (Table 4). Because nearly all of these systematic tub1 mutations alter benomyl sensitivity, it appears that microtubules are exquisitely sensitive to changes in Tub1p sequence.

Figure 1.

Phenotypes of TUB1 and TUB2 Ala-scan mutants. The positions of the mutations and their resulting phenotypes are shown for both TUB1 (top) and TUB2 (bottom). The allele number of each mutation is shown, and the altered residues are in bold. SS, benomyl-supersensitive; R, benomyl-resistant; WT, wild-type; CS, cold-sensitive; TS, heat-sensitive; SLOW, slow growth at all temperatures. The TUB2 Ala-scan data is reproduced from Reijo et al. (1994), and phenotype definitions are adapted to be identical to those used in describing the TUB1 mutants.

Figure 2.

Growth of tub1 mutants at various temperatures. The growth of each mutant was compared with the growth of the wild-type control across a range of temperatures. Growth ranges from wild-type growth (heavily shaded) to no growth (unshaded).

Table 4.

tub1 and tub1 tub3 mutant phenotypes

Category of benomyl sensitivitya	tub1 allele	tub1 phenotypeb	tub1 tub3Δ phenotypeb
1	801	BenSS, Cs, Ts	SL
	809	BenSS, slow	SL
	813	BenSS, slow	SL
	814	BenSS, slow	SL
	817	BenSS	SL
	822	BenSS, slow	SL
	824	BenSS, Cs	SL
	827	BenSS, slow	SL
	829	BenSS, slow	SL
	833	BenSS	BenSS, slow
	834	BenSS, slow	SL
	836	BenSS, Cs, Ts	SL
2	810	BenSS	SL
	816	BenSS, Cs	SL
	818	BenSS, Cs	SL
	819	BenSS	BenSS, slow
	830	BenSS	SL
	837	BenSS	SL
	845	BenSS, slow	SL
	846	BenSS, Cs, Ts	SL
	849	BenSS, slow	SL
	853	BenSS, slow	SL
3	803	BenSS	BenSS, slow
	804	BenSS	BenSS, Cs
	805	BenSS	BenSS, slow
	807	BenSS	BenSS, slow
	812	BenSS	BenSS, Ts
	821	BenSS	SL
	831	BenSS, Cs	BenSS, slow
	838	BenSS	SL
	842	BenSS, Cs, Ts	SL
	844	BenSS, Cs, Ts	SL
	847	BenSS, slow	SL
	848	BenSS, slow	SL
	850	BenSS	BenSS, slow
	851	BenSS	BenSS, slow
	852	BenSS	BenSS
4	TUB1	WT	BenSS
	811	WT	BenSS
	815	WT	BenSS
	835	WT	BenSS
	843	WT	BenSS, Cs
5	806	BenR	BenSS
	825	BenR	BenSS
	826	BenR	BenSS
	832	BenR	BenSS
	839	BenR	BenSS
	841	BenR	BenSS, slow

^a1, more sensitive than tub3Δ; 2, sensitivity equal to tub3Δ; 3, less sensitive than tub3Δ but more sensitive than wild type; 4, sensitivity equal to wild type; 5, less sensitive than wild type (benomyl-resistant). 

^bBenR, benomyl resistant; BenSS, benomyl supersensitive; Cs, cold-sensitive; SL, synthetic lethal; slow, slow growth at all temperatures; Ts, heat-sensitive; WT, wild-type. 

Synthetic Lethality with Deletions of TUB3

To determine the phenotype of each tub1 allele in the absence of any wild-type α-tubulin, each was combined with a deletion of the minor α-tubulin gene TUB3. The resulting phenotypes are shown in Table 4. None of the lethal tub1 mutants was rescued by removing TUB3. Remarkably, more than half of the viable tub1 mutants (26 of 47) were synthetically lethal in combination with tub3Δ. Furthermore, many of the remaining viable tub1 tub3Δ double mutants were severely growth impaired. TUB3 obviously plays a vital role in the microtubule function of most of the tub1 mutants.

Categorizing the tub1 mutants according to their degree of benomyl supersensitivity was a very good predictor of the viability of the tub1 tub3Δ double mutant. Alongside each mutant allele in Table 4 are any temperature and/or cold sensitivities produced by that allele, as well as its tub3Δ double mutant phenotype. Almost without exception, the most benomyl-supersensitive and growth-impaired tub1 alleles are also the alleles that are synthetically lethal with tub3Δ. The most parsimonious explanation for this observation is that the phenotypic effect of removing TUB3 from tub1 strains is merely additive, so that the most severe tub1 alleles will not be able to tolerate it, whereas the less severe alleles will. The exceptions to this rule may indicate TUB1- or TUB3-specific functional regions or may indicate regions where a particular residue is required, but only in a subset of α-tubulin subunits. For example, the tub1-844 mutant, although just slightly more benomyl-supersensitive than the wild-type, does not survive in the absence of TUB3. The residue altered by this allele may be crucial for microtubule function, but the few monomers encoded by TUB3 are sufficient to allow microtubule function.

Microtubule and Nuclear Phenotypes of Cold-sensitive tub1 Mutants

The cold-sensitive tub1 mutants were further examined by visualizing microtubules themselves with indirect immunofluorescence. Previously, tub1 mutants were found to fall in three classes. Class 1 mutants had very few or no detectable microtubules, class 2 mutants had extra and/or longer microtubules, and class 3 mutants had relatively normal numbers of microtubules, sometimes disorganized. (Schatz et al., 1988). This system provided a convenient classification scheme for organizing the current set of tub1 mutants as well.

The microtubules were observed after fixing cells grown at the nonpermissive temperature. In general, the observed phenotypes agreed very well with the tub1 mutant phenotype categories defined by Schatz et al. (1988). Several of the mutants displayed reduced or no staining at the restrictive temperature, indicating that they have impaired microtubule formation or stability. Many mutants, other than having an absence of long spindles, as would be predicted from the defect in nuclear division seen by DAPI staining (see below), displayed no other distinct microtubule phenotypes. One mutant, tub1-831, has brighter, more numerous, and longer than normal extranuclear microtubules throughout the cell cycle. This mutant phenotype is similar to the class 2 mutant microtubule phenotype described by Schatz et al. (1988). Table 5 shows the microtubule phenotypes of each of the tub1 mutants.

Table 5.

Microtubule phenotypes of tub1 mutants and tub1 tub3Δ double mutants

tub1 mutant	Microtubule class	Growth phenotype	tub1 tub3Δ mutant	Microtubule class	Growth phenotype
800 (wild-type)	3	WT	800 tub3Δ	3	BenSS
801	3	BenSS, Cs, Ts	803 tub3Δ	1	BenSS, slow
806	3	BenR	805 tub3Δ	3	BenSS, slow
809	1	BenSS, slow	806 tub3Δ	3	BenSS
813	3	BenSS, slow	807 tub3Δ	1	BenSS, slow
814	3	BenSS, slow	812 tub3Δ	3	BenSS, Ts
822	3	BenSS, slow	819 tub3Δ	3	BenSS, slow
827	1	BenSS, slow	825 tub3Δ	3	BenSS
829	3	BenSS, slow	831 tub3Δ	3	BenSS, slow
831	2	BenSS, Cs	833 tub3Δ	3	BenSS, slow
834	1	BenSS, slow	850 tub3Δ	3	BenSS, slow
845	3	BenSS, slow	851 tub3Δ	1	BenSS, slow
846	3	BenSS, Cs, Ts	852 tub3Δ	3	BenSS
847	3	BenSS, slow
848	3	BenSS, slow
849	3	BenSS, slow
853	3	BenSS, slow

Microtubules from mutant cells grown for two generations at 11°C were visualized using indirect immunofluorescence. Intensity was scored in comparison with wild type (TUB1-800), which was included in every experiment. Mutants are divided into three categories according to their microtubule phenotypes: 1) very diminished or no microtubules, 2) excess microtubules, and 3) normal microtubules, sometimes fainter than usual. Growth phenotypes are shown for comparison, and abbreviations are as listed in Table 4. 

Many of the cold-sensitive mutants were examined at the restrictive temperature (11°C) for nuclear phenotypes resulting from disrupted microtubules. Two primary microtubule defects, nuclear migration and nuclear division, were quantified using the DNA dye DAPI to visualize nuclei, and cell cycle progression was assessed by determining the distribution of bud sizes. Nuclear migration requires extranuclear microtubules (Huffaker et al., 1988). In wild-type cells, nuclear migration happens early in the cell cycle, so large-budded cells are almost never observed without the nucleus at or extended through the bud neck. The other microtubule defect that was visualized with DAPI staining is in nuclear division, a process requiring intranuclear microtubules (Huffaker et al., 1988). In tubulin mutants, when chromosomes do not separate because of a nonfunctional spindle, the nucleus does not divide. This is easily visualized as large-budded cells containing a single DAPI-staining region, rather than the two distinct DAPI-staining regions seen in wild-type large-budded cells.

The tub1 mutants accumulate large-budded cells to varying degrees and show a variety of nuclear phenotypes, shown in Figure 3. Some mutants become dramatically worsened at 11°C; whereas some are equally affected at both temperatures. Some alleles produce defects predominantly in either nuclear division or nuclear migration, whereas some alleles affect both processes to the same extent. Analysis of these phenotypes leads to several general conclusions. First, the accumulation of large-budded cells, although not absolute, is significant in most of the tub1 mutants, probably arising from a delay in mitosis. Second, the tub3Δ mutant has both a nuclear migration and a detectable nuclear division defect that is more severe at 11°C. This ability to distinguish a tub3Δ strain from the wild type demonstrates the sensitivity of this assay and reveals two new, albeit subtle, phenotypes for the tub3Δ mutant. Third, the tub1 tub3Δ double mutants have generally more severe defects in nuclear migration than tub1 mutants, in contrast with nuclear division, which is affected to the same degree. Last, the defects in nuclear division resulting from the tub1 Ala-scan mutations are more dramatic than the defects in nuclear migration, in concordance with the results obtained from analysis of the tub2 Ala-scan mutants.

Figure 3.

Nuclear phenotypes and cell cycle progression of tub1 mutants. Large-budded cells were examined both by ascertaining their frequency in a growing population of cells and by staining their nuclei with DAPI. Cells were examined both at 25°C and after a shift to 11°C for two generations. In A, the percentage of large-budded cells with an undivided nucleus was determined; in B, the percentage of large-budded cells with a nucleus away from its normal position near the bud neck was determined; and in C, the percentage of large-budded cells (bud diameter greater than one-half of the mother cell's body) in the population was determined.

Comparison of tub1 and tub2 Mutant Phenotypes

Because microtubules are made of heterodimers of α- and β-tubulin, it is worthwhile to compare the set of TUB1 Ala-scan mutants and the set of TUB2 Ala-scan mutants (Reijo et al., 1994). It should be noted that direct comparisons between tub1 and tub2 mutant phenotypes are complicated by the existence of a second α-tubulin gene (TUB3). Nonetheless, there are several notable comparisons. First, both the TUB1 and TUB2 systematic mutageneses produced a few dominant lethal alleles. There were more recessive lethal alleles in TUB2, which is not surprising, given that TUB2 is the sole β-tubulin gene, whereas TUB3 provides a nontrivial fraction of the cell's α-tubulin. Second, there are more tub2 benomyl-resistant mutants. Mutations that simultaneously result in benomyl resistance and cold sensitivity were found only in TUB2. In TUB1, all of the benomyl-resistant mutants show wild-type growth at all temperatures. Finally, a much higher percentage of tub1 mutants are benomyl supersensitive, leaving only three tub1 mutants with a normal growth phenotype, many fewer than the number of normally growing tub2 mutants.

The results of comparing analogous mutations in TUB1 and TUB2 are shown in Figure 1. Although the identical mutation was rarely made, it is nonetheless notable how dissimilar the results are across the entire length of the proteins. Six of the tub1 mutations (tub1-801, -808, -809, -824, -847, and -853) are absolutely identical to their cognate tub2 mutation, yet none of these shares the same phenotype with its tub2 counterpart. There are three lethal tub1 mutations that seem to be in essential regions shared between the two proteins (see tub1-802, -820, and -828), but in other cases (tub1-823 and tub2-454) the exact counterpart of a lethal mutation in one gene has a viable phenotype in the other gene. Furthermore, the dominant lethal TUB1 mutations are in the N-terminal half of the protein, whereas the dominant lethal TUB2 mutants are in the C-terminal half. This illustrates what appears to be an essentially complete functional divergence between the two components of the tubulin heterodimer, even though their three-dimensional structure is nearly identical and their primary sequence is very similar. This seems a natural consequence of the polarity of both the tubulin dimer and the microtubule and of the different involvement of α- and β-tubulin in the interaction with other cellular components.

Structure–Function Relationships

The amino acids altered by tub1 and tub2 systematic mutations were mapped onto a three-dimensional model (see MATERIALS AND METHODS) of the yeast tubulin dimer. Most of the mutated residues are distributed relatively evenly across the protein surface. Figure 4 illustrates this distribution for Tub1p; the distribution of mutations on Tub2p is similar. The longitudinal interfaces and the microtubule-interior face of each monomer have regions (10–15 Å in diameter) that are devoid of charged residues. A small fraction of the mutated residues are buried in the structure, including Tub1p residues R65, D70, T146, E169, D206, and R321 and Tub2p residues R62, D67, E123, E128, D203, and R318.

Figure 4.

Distribution of alanine-scanning mutations in Tub1p. Side chains of amino acids altered by tub1 mutations are colored as in Figure 1: Green, wild-type; red, lethal; blue, cold-sensitive; orange, slow growth; gray, benomyl-supersensitive; purple, benomyl-resistant. Tub2p shown as a backbone trace, and Tub1p is shown as space-filled atoms. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow in Tub2p (Nogales et al., 1999). Orientation axes: + and − show microtubule orientation; H3 and M show lateral sides marked by helix H3 α-helix and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using RASMOL (Sayle and Milner-White, 1995).

The distribution of mutations in different phenotypic classes was examined. Tubulin mutations often result in the unusual phenotype of cold sensitivity. As shown in Figure 5, the distribution of the cold-sensitive alleles is striking; all these alleles, in both Tub1p and Tub2p, lie either at or near the lateral contacts between protofilaments or in longitudinal contacts between tubulin monomers along the protofilament. In other words, virtually every cold-sensitive tubulin Ala-scan mutation is physically in a position to disrupt the polymerized microtubule structure by altering tubulin–tubulin contacts. This is consistent with the fact that microtubules are known to be cold-sensitive structures.

Figure 5.

Mutations resulting in cold sensitivity are located in areas of the tubulin protein involved in longitudinal or lateral contacts in the microtubule polymer. (A) View from the outside of the microtubule. (B) View from the side of the protofilament. The side chains of amino acids mutated in cold-sensitive alleles (red) and temperature- and cold-sensitive alleles (orange) are shown on Tub2p (top) and Tub1p (bottom) of the yeast tubulin heterodimer. These amino acids are labeled by their allele number. Lateral interaction elements, α-helix H3 and M-loop, are shown in yellow (Nogales et al., 1999). GTP and GDP are shown in green. Adjacent monomers of the protofilament are shown in lighter gray. Orientation axes: + and − show microtubule orientation; lat_H3 and lat_M show lateral sides marked by helix H3 and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

The mutations producing a benomyl-resistant phenotype were mapped onto the tubulin model. Benomyl-resistant mutations were split into two classes; five tub2 mutants were resistant to very high concentrations of benomyl (75 or 80 μg/ml), and the remainder were resistant only to lesser concentrations. In wild-type Tub2p each of the five (Figure 6, orange) contain component charged residues (123, 197, 198, and 318) in a pocket relatively deep in the tubulin structure (unusual for charged residues). This pocket, situated between the β-sheet S1–S6, helix H8 and the core helix H7, is also the site of several other highly benomyl-resistant mutations, tub2-201 (L253V), tub2-104 (R241H), and the benomyl-dependent tub2-150 (T238A) (Figure 6, purple; Thomas, 1984; Thomas et al., 1985; Machin et al., 1995). It seems possible, although we have no more direct evidence, that this pocket might be associated with either benomyl binding or a conformational response to benomyl binding. Consistent with this possibility, residue 198 is found to be changed in many benomyl-resistant mutations in the β-tubulin-encoding genes of a variety of species other than S. cerevisiae (e.g., Fujimura et al., 1992; Jung et al., 1992; Buhr and Dickman, 1994; Park et al., 1997).

Figure 6.

Location of benomyl-resistant tub1 and tub2 alleles. (A) View from the outside of the microtubule. (B) View from the side of the protofilament. (C) Cutaway view of the core of β-tubulin, with the interior-facing loop (residues 24–62) removed, β-sheet S1–S6 is shown in orange, and the core helix H7, helix H8, and the intervening T7 loop are shown in red (Nogales et al., 1998b). The side chains of amino acids mutated in alleles resistant to only 40 μg/ml benomyl (red) and alleles resistant to at least 80 μg/ml benomyl (orange) are shown on Tub2p and Tub1p of the yeast tubulin heterodimer, labeled by their allele number. The amino acids mutated in tub2 alleles 431 (E194A, H195A, and E198A), 432 (E194A and D197A), and 433 (E198A) are individually labeled by their residue name and number. The amino acids mutated in tub2 alleles 201, 104, and 150 (Thomas et al., 1985; Machin et al., 1995) are shown in purple. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow (Nogales et al., 1999). GTP and GDP are shown in green. Adjacent monomers of the protofilament are shown in lighter gray. Orientation axes: + and − show microtubule orientation; lat_H3 and lat_M show lateral sides marked by helix H3 α-helix and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

The second class of benomyl-resistant alleles includes both tub1 and tub2 alleles. Whereas these benomyl-resistant tub2 mutations are distributed across Tub2p, all of the benomyl-resistant tub1 alleles except one cluster to a face of Tub1p near the lateral protofilament contact but exposed to the outer surface of the microtubule. Because an increased level of α-tubulin can cause benomyl resistance (Schatz et al., 1986b), it is a formal possibility that these mutations might increase the quantity of functional α-tubulin. Alternatively, they might disrupt the binding of a protein that acts to destabilize microtubules. For example, deletion of the DYN1 or KIP1 genes encoding microtubule motor proteins results in benomyl resistance and increased microtubule length (Cottingham and Hoyt, 1997). Interestingly, several benomyl-resistant mutations in both tub1 and tub2 map to loops that would be in the interior of an assembled microtubule (Figure 6, see tub1-806 and tub2-410 and -411).

Residues Affecting Bim1p Binding

We previously described a microtubule-binding protein, Bim1p, and demonstrated that it bound to Tub1p in the two-hybrid system and decorated microtubules in vivo. All of the tub1 Ala-scan alleles were tested for binding to Bim1p in the two-hybrid system, and tub1 mutations that disrupted binding were found to cluster at the C terminus of Tub1p (Schwartz et al., 1997). The tub1 mutations that disrupt Bim1p binding are mapped on the structure of α-tubulin in Figure 7. Most of these mutations cluster in a region facing outward in the assembled microtubule; however, there are several (tub1-809, -813, -842, and -846) that were located at the intradimer interface. Perhaps an α–β dimer must be formed to achieve a positive two-hybrid interaction with Bim1p. Two-hybrid interactions were not tested between Bim1p and the Tub2p Ala-scan mutants, because the ectopic expression of Tub2p from these plasmids was lethal, as expected (Burke et al., 1989; Weinstein and Solomon, 1990).

Figure 7.

Two-hybrid interaction footprint of Bim1p on Tub1p. Bim1p was tested for interaction with each of the Tub1p mutant proteins using the two-hybrid system. Those tub1 mutations that abolished the two-hybrid interaction are shown in red; those tub1 mutations that still interacted are shown in blue (Schwartz et al., 1997). Structure drawn using MIDAS (Ferrin et al., 1988).

A similar approach has been used to delineate the interaction footprint of another Tub1p-binding protein, Alf1p (Feierbach et al., 1999), which lies at a site overlapping but not identical to the Bim1p footprint at the C terminus of Tub1p, on the outside face of the microtubule. Motor proteins, such as dynein and kinesin, have also been shown to bind β-tubulin, also at the C terminus (Goldsmith et al., 1995; Hirose et al., 1995; Hoenger et al., 1995).

Double Mutant Analysis

Another approach to examine interactions between two proteins is by differential genetic interactions. We tested the genetic interactions between each of the tub1 Ala-scan mutants and a benomyl-resistant mutant, tub2-201. The tub2-201 mutation confers a high level of benomyl resistance but no other discernible phenotypes (Thomas, 1984). When sequenced, tub2-201 contained a Leu to Val change at position 253, which maps to the interior side of helix H8 at the intradimer interface (see Figures 6 and 8). DBY7830, which contains tub2-201 back-crossed into the DBY6654 strain background (Schwartz et al., 1997), was crossed to each of the tub1 Ala-scan mutants. The double mutants displayed a range of phenotypes, from wild-type growth to synthetic lethality. On media containing benomyl, the double mutants generally showed additive effects; i.e., they displayed the benomyl resistance of tub2-201; but somewhat diminished commensurate with the degree of benomyl sensitivity of the single tub1 mutant. The tub1 mutations that were synthetically lethal in combination with tub2-201 are shown in Figure 8. These tub1 alleles (-809, -813, -814, -845, and -853) cluster at the intradimer interface. This suggests that tub2-201, although nearly wild-type in phenotype, may cause a distortion of its intradimer interface. When tub2-201 is combined with tub1 mutations at the intradimer interface, the α–β dimer interaction may become so unstable as to cause death. In support of this hypothesis, tub1-814 contains a mutant residue 106 that is known to be important for stabilizing the intradimer interaction in yeast (Vega et al., 1998).

Figure 8.

tub1 alleles that are synthetically lethal with tub2-201 localize to the intradimer interface. Amino acids mutated in these tub1 alleles (red) are shown on Tub1p (bottom). Tub2p is partly shown above with the tub2-201 mutant residue shown in magenta. Lateral interaction elements, α-helix H3 and M loop, are shown in yellow (Nogales et al., 1999). GTP is shown in green. The view is from the side of the heterodimer. Orientation axes: + and − show microtubule orientation; lat_H3 and lat_M show lateral sides marked by helix H3 and M loop, respectively; in and out represent the inside and the outside of the microtubule, respectively. Structure drawn using MOLSCRIPT (Kraulis, 1991).

Conclusion

The TUB1 mutations described here complete the systematic mutagenesis of the genes encoding the major subunits of the yeast cytoskeleton. Although there are two α-tubulin-encoding genes, TUB1 and TUB3 are so similar that it is likely that it will be possible to study virtually all structure–function relationships with associated proteins using the mutations already in hand. Mapping of the mutations to the surface will provide, for tubulin, genetic tools for structure–function analysis analogous to those that have served so well in the case of actin, in which mutations derived from a similar systematic mutagenesis scheme have served to connect residues on the actin three-dimensional structure to binding regions for a great variety of ligands. To illustrate this, the new tub1 mutants described here have allowed us to understand better the interaction of α- with β-tubulin and α-tubulin with Bim1p and to suggest the location of the benomyl-binding site by placing tub2 mutations on the structure.