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Structure-function relationships in yeast tubulins.

ABSTRACT: A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-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 tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-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 beta-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 alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta 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.

SUBMITTER: Richards KL

PROVIDER: S-EPMC14891 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Structure-function relationships in yeast tubulins.

Richards K L KL Anders K R KR Nogales E E Schwartz K K Downing K H KH Botstein D D

Molecular biology of the cell 20000501 5

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-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 tub3Delta, supporting the idea that benomyl supersensi ...[more]

PMID: 10793159

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Project description:The crystal structures of the Ca(2+)- and H(+)-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure-function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC(50) for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the K(m) for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E(2) conformation of the ATPase towards the E(1) conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383-2392, 2007) led to a dramatic increase in the apparent stoichiometry of H(+) transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.

Project description:PurposeTo investigate the relationship between longitudinal changes in macular thickness measurements from OCT and changes in central visual field (VF) in patients with glaucoma with central or advanced damage at baseline.DesignLongitudinal cohort study.ParticipantsA total of 116 eyes with ≥3 years of follow-up and ≥5 macular OCT images and central 10° VF tests were selected.MethodsOCT superpixels and VF locations were matched correcting for retinal ganglion cell (RGC) displacement. Superpixel thickness and VF total deviation (TD) values, in both logarithmic and linear scales, were averaged within 3 eccentricities (3.4°, 5.6°, and 6.8°) and superior and inferior hemiretinas and hemifields. We estimated pointwise TD rates of change and rates of change at superpixels for full macular thickness (FMT), ganglion cell complex (GCC), ganglion cell inner plexiform layer (GCIPL), and ganglion cell layer (GCL). Correlation of structure-function (SF) rates of change was investigated with parametric tests. We compared the proportion of worsening and positive slopes for superpixels and VF test locations (negative vs. positive rates of change with P < 0.05) throughout the follow-up period. Permutation analyses were used to control specificity.Main outcome measuresMagnitude of correlation between structural and functional rates of change and proportion of worsening and positive slopes as a function of follow-up time.ResultsThe median (interquartile range) follow-up and number of exams were 4.2 (3.7-4.6) years and 8 (7-9), respectively. The highest correlation of change rates was observed at 3.4° and 5.6° eccentricities (r = 0.24, 0.41, 0.40, and 0.40 for FMT, GCC, GCIPL, and GCL for 3.4° eccentricity and r = 0.28, 0.32, 0.31, and 0.32 for FMT, GCC, GCIPL, and GCL for 5.6° eccentricity, respectively). Although GCC measures demonstrated the highest overall longitudinal SF correlations, the differences were not statistically significant. Significant structural worsening was more frequently detected than functional deterioration at 3- and 5-year time points (P < 0.025). Permutation analyses also confirmed this finding.ConclusionsCorrelations between central structural and functional rates of change were weak to fair in this cohort. Structural changes were detected more frequently than functional changes. Measurements of both structure and function are required for optimal detection of central progression.

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Structure-function relationships in yeast tubulins.

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Structure-function relationships in yeast tubulins.

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