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Analyses of the effects of all ubiquitin point mutants on yeast growth rate.

ABSTRACT: The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent-inaccessible core of ubiquitin, all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that, for these mutants, subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.

SUBMITTER: Roscoe BP

PROVIDER: S-EPMC3615125 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Analyses of the effects of all ubiquitin point mutants on yeast growth rate.

Roscoe Benjamin P BP Thayer Kelly M KM Zeldovich Konstantin B KB Fushman David D Bolon Daniel N A DN

Journal of molecular biology 20130130 8

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defec ...[more]

PMID: 23376099

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Project description:BackgroundControl of growth rate is mediated by tight regulation mechanisms in all free-living organisms since long-term survival depends on adaptation to diverse environmental conditions. The yeast, Saccharomyces cerevisiae, when growing under nutrient-limited conditions, controls its growth rate via both nutrient-specific and nutrient-independent gene sets. At slow growth rates, at least, it has been found that the expression of the genes that exert significant control over growth rate (high flux control or HFC genes) is not necessarily regulated by growth rate itself. It has not been determined whether the set of HFC genes is the same at all growth rates or whether it is the same in conditions of nutrient limitation or excess.ResultsHFC genes were identified in competition experiments in which a population of hemizygous diploid yeast deletants were grown at, or close to, the maximum specific growth rate in either nutrient-limiting or nutrient-sufficient conditions. A hemizygous mutant is one in which one of any pair of homologous genes is deleted in a diploid, These HFC genes divided into two classes: a haploinsufficient (HI) set, where the hemizygous mutants grow slower than the wild type, and a haploproficient (HP) set, which comprises hemizygotes that grow faster than the wild type. The HI set was found to be enriched for genes involved in the processes of gene expression, while the HP set was enriched for genes concerned with the cell cycle and genome integrity.ConclusionA subset of growth-regulated genes have HFC characteristics when grown in conditions where there are few, or no, external constraints on the rate of growth that cells may attain. This subset is enriched for genes that participate in the processes of gene expression, itself (i.e. transcription and translation). The fact that haploproficiency is exhibited by mutants grown at the previously determined maximum rate implies that the control of growth rate in this simple eukaryote represents a trade-off between the selective advantages of rapid growth and the need to maintain the integrity of the genome.

Dataset Information

Analyses of the effects of all ubiquitin point mutants on yeast growth rate.

Publications

Analyses of the effects of all ubiquitin point mutants on yeast growth rate.

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