Systematic mutational analysis of the yeast beta-tubulin gene.

Reijo, ReneeA .; Cooper, Emily; Beagle, G J; Huffaker, Tim C.

doi:10.1091/mbc.5.1.29

Cited by 100 publications

(104 citation statements)

References 28 publications

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“…(46) In yeast β tubulin, the double mutant E194A/D197A caused benomyl resistance but cells were viable. (47) In yeast α tubulin, a triple mutant containing these two plus H198A was recessive lethal, but it is not known which mutation caused the defect. (48) This conclusion that the amino acids involved in binding and hydrolysis of GTP are highly conserved is a new one for FtsZ and tubulin.…”

Section: Amino Acids Highly Conserved From Ftsz To Tubulin Are Those mentioning

confidence: 99%

Evolution of the cytoskeleton

2007

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SummaryThe eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40−50% sequence identity across different bacterial and archaeal species. Here I suggest that this represents the limit of divergence that is consistent with maintaining their functions for cytokinesis and cell shape. Previous analyses have noted that tubulin and actin are highly conserved across eukaryotic species, but so divergent from their prokaryotic relatives as to be hardly recognizable from sequence comparisons. One suggestion for this extreme divergence of tubulin and actin is that it occurred as they evolved very different functions from FtsZ and MreB. I will present new arguments favoring this suggestion, and speculate on pathways. Moreover, the extreme conservation of tubulin and actin across eukaryotic species is not due to an intrinsic lack of variability, but is attributed to their acquisition of elaborate mechanisms for assembly dynamics and their interactions with multiple motor and binding proteins. A new structure-based sequence alignment identifies amino acids that are conserved from FtsZ to tubulins. The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP. Historical introduction Discoveries of the prokaryotic cytoskeletonBefore 1990, the cytoskeleton was thought to have evolved only in eukaryotes. Relatives or homologs of actin, tubulin or intermediate filaments were unknown in bacteria or archaea. There were sporadic reports of candidates for bacterial actin and tubulin in the 1970s and 1980s but, apart from some intriguing images of microtubules in certain bacteria,(1) these turned out to be wrong and/or were not followed up.The first suggestions of a bacterial homolog of tubulin appeared in 1992. Three groups independently discovered that the bacterial cell division protein FtsZ bound and hydrolyzed GTP, as does tubulin, and had a seven-amino-acid sequence, GGGTGTG, virtually identical to the "tubulin signature sequence".(2-4) Mukherjee and Lutkenhaus(37) then extended the sequence alignment to find a dozen additional amino acids that were completely conserved in FtsZ and in α, β and γ tubulins. They also showed that FtsZ assembled in vitro into filamentous structures. Erickson et al. (5) found that FtsZ assembled into protofilaments that could adopt two conformations, straight and curved, similar to the straight protofilaments of the microtubule wall and the tubulin rings that peel away from microtubules during disassembly. Any question of homology was resolved when tubulin and FtsZ were found to have virtually identical structures at the level of protein folding.(6,7)If one looks for bacterial relatives of tubulin or actin using a simple computer search for sequence similarity, nothing will be found. In 1992 Bork et al.(8) used a sophisticated structurebased sequence alignment, and they discovered that bacteria did have genes related to actin. Actin is...

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Section: Amino Acids Highly Conserved From Ftsz To Tubulin Are Those mentioning

confidence: 99%

Evolution of the cytoskeleton

2007

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“…In yeast the mutation of K59 and R63 to alanines is lethal in ␣-tubulin (Richards et al, 2000), whereas it is coldsensitive in ␤-tubulin (Reijo et al, 1994). This residue could be important for tubulin folding.…”

Section: Mipar63mentioning

confidence: 99%

“…Clustered charged-to-alanine scanning mutagenesis has been a highly successful approach for creating conditionally lethal alleles of cytoskeletal proteins (including ␣-and ␤-tubulin) and for defining regions essential to the functioning of those proteins (Wertman et al, 1992;Reijo et al, 1994;Richards et al, 2000, and references therein). The principle behind this approach is that charged regions tend to be on the outside of proteins and replacement of charged amino acids with alanines often alters regions of interprotein interactions without causing gross structural changes.…”

Section: Introductionmentioning

confidence: 99%

Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

Jung

Prigozhina

Oakley

et al. 2001

MBoC

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We have created 41 clustered charged-to-alanine scanning mutations of the mipA, ␥-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of ␥-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that ␥-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of ␥-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

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“…Relaxation of the M loop conformation, particularly in the ␤-subunit, may enable the ␤/␤ lateral interaction to accommodate to the probable hydrolysis-dependent conformational change in H3, thus mitigating its destabilizing effect. It is interesting that many cold-sensitive, charged-to-alanine mutants of yeast ␣-and ␤-tubulins map to their respective M loops, H3 helices, and H1-S2 loops, the major contributors to lateral, interprotofilament contact (43,44). Second, provision of additional hydrophobic interactions at the lateral surface of the ␣-chain would strengthen interprotofilament interaction at low temperature and explain the greater entropic control of the polymerization of the fish tubulins.…”

Section: Cold Adaptation Of Microtubule Assembly Via Small Numbers Ofmentioning

confidence: 99%

Cold Adaptation of Microtubule Assembly and Dynamics

Detrich¹,

Parker²,

Williams³

et al. 2000

Journal of Biological Chemistry

143

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The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (؊1.8°C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0°C to ϳ0.9 mg/ml (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790 -2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5°C) and high (25°C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37°C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 ␣-tubulins and 4 ␤-tubulins from the yellowbelly rockcod Notothenia coriiceps and 4 ␣-tubulins and 2 ␤-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the ␣-and ␤-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in ␣; S280G and A285S in ␤) were present in the S7-H9 interprotofilament "M" loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the ␣-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (␣:S187A in helix H5 and ␤:Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.The capacity of the cytoplasmic tubulins of Antarctic fishes to form microtubules at temperatures as low as Ϫ1.8°C, the freezing point of the seawater habitat of these fish, is remarkable. The critical concentration for polymerization at 0°C of purified, MAP 1 -free brain tubulin is ϳ0.9 mg/ml (1-3), which approximates the values observed for mammalian brain tubulins at 37°C (1, 4). Unlike mammalian microtubules, however, the cytoplasmic microtubules of Antarctic fishes are very stable and exhibit slow dynamics at cold temperatures (5, 6). Evidently, both the capacity to polymerize at low critical concentration and the slow exchange of tubulin dimers into and out of microtubule polymers are properties intrinsic to the tubulin subunits of these fishes. These unusual functional characteristics must be explicable by structural alterations to the primary sequences and/or by changes in the posttranslational modifications of the tubulin chains that, alone or in concert, modify the quaternary interactions between dimers in a microtu...

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Systematic mutational analysis of the yeast beta-tubulin gene.

Cited by 100 publications

References 28 publications

Evolution of the cytoskeleton

Evolution of the cytoskeleton

Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

Cold Adaptation of Microtubule Assembly and Dynamics

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