Eugenol does not induce leakage of liposomes made of total yeast lipids or inhibit protein synthesis.

<p>(A) Release of carboxyfluorescein from large unilamellar vesicles (LUVs) over a series of eugenol concentrations (0.003 to 1.6 mg/ml, bottom axis) was compared to 100% release from liposome exposed to Triton-X 100. <i>Echinacea purpurea</i> extract was used as a positive lysis control over a concentration gradient of 0.01 to 5% (top axis). Data correspond to the mean % leakage values (±SD) of three independent experiments. (B) Yeast exposed to subinhibitory (0.21 mg/ml) or inhibitory (0.27 mg/ml) concentrations of eugenol do not have significantly decreased β-galactosidase activity in comparison to the untreated control. In contrast, the inhibitor of protein translation, cycloheximide, significantly reduced β-galactosidase activity in the assay. These observations indicate that eugenol does not reduce efficiency of translation in yeast as would be expected for compounds that perturb the intracellular pool of amino acids. The values are expressed as mean ±SD of triplicates, difference between treatment and untreated control are indicated as p<0.05 (*) and p<0.01 (**).</p

Nearly half of eugenol-sensitive strains have deletions in genes involved in aromatic and branched-chain amino acid synthesis or uptake.

<p>(A) The haploid non-essential yeast gene deletion array was subjected to a subinhibitory concentration of eugenol. Colony size reduction was used to detect sensitivity. The mutants most sensitive to eugenol were clustered according to the cellular processes in which their deleted genes participated. (B) Eugenol-sensitive strains identified by GDA were verified by drop out plates. Wild type and eight randomly selected gene deletion mutant strains that were eugenol-sensitive based on GDA analysis were 10-fold serially diluted and spotted on solid YPD medium with a subinhibitory concentration (0.18 mg/ml) of eugenol or without eugenol (control). The plates were incubated at 30°C for 1–2 days and then photographed. All deletion mutants selected exhibited increased sensitivity to eugenol, providing verification of the GDA analysis.</p

Auxotroph supplement assay shows that eugenol inhibits the functions of aromatic and branched-chain amino acid transporters.

<p>Strains of <i>S. cerevisiae</i> from GDA library were selected with gene deletions in the tryptophan, phenylalanine, tyrosine (A) or isoleucine (B) biosynthesis pathways. Cultures were 10-fold serially diluted and spotted on synthetic medium supplemented with tryptophan, phenylalanine, tyrosine and isoleucine, either containing a subinhibitory concentration of eugenol (0.18 mg/ml) or without eugenol (control). The plates were incubated at 30°C for 1–2 days and then photographed.</p

Phenotypic suppression assay shows that Tat1p and Gap1p permeases are targets of eugenol in the yeast cytoplasmic membrane.

<p>Overexpression constructs of four permeases, <i>BAP2, GAP1</i>, <i>TAT1</i> and <i>TAT2</i> were transformed separately into the <i>aro1Δ</i> strain. Transformants were grown overnight in SC-ura medium, diluted 1∶300 and then added to each well of a sterile microtiter plate with or without 0.18 mg/ml eugenol. Growth of the <i>aro1Δ</i> transformants containing the overexpression constructs were compared to one with a control plasmid by measuring the optical density of cells in each well (OD₆₀₀). The values are expressed as mean (±SD, n = 3) and significant differences between treatment and plasmid control are indicated as <i>P<0.05</i> (*) and <i>P<0.01</i> (**) based on Student T-test. Inset: eugenol is structurally similar to aromatic amino acids and is synthesized in plants via the phenylpropanoid pathway from phenylalanine.</p

List of eugenol-sensitive gene deletion mutants from GDA analysis that showed greater than 70% reduction in colony size.

<p>List of eugenol-sensitive gene deletion mutants from GDA analysis that showed greater than 70% reduction in colony size.</p

Significant protein clusters and GO term enrichment analysis results found in each of the four non-<i>cerevisaie</i> species.

<p>Significant protein clusters and GO term enrichment analysis results found in each of the four non-<i>cerevisaie</i> species.</p

Proteins which experience a lower or higher number of changes in PPIs in the real data compared to the simulated interactomes.

<p>Proteins which experience a lower (A) or higher (B) number of changes in inferred PPIs in the real data in comparison to the simulated interactomes. Each protein’s real γ is plotted in red and the range of γ observed in the null model are plotted in black.</p

Evolution of protein-protein interaction networks in yeast

<div><p>Interest in the evolution of protein-protein and genetic interaction networks has been rising in recent years, but the lack of large-scale high quality comparative datasets has acted as a barrier. Here, we carried out a comparative analysis of computationally predicted protein-protein interaction (PPI) networks from five closely related yeast species. We used the Protein-protein Interaction Prediction Engine (PIPE), which uses a database of known interactions to make sequence-based PPI predictions, to generate high quality predicted interactomes. Simulated proteomes and corresponding PPI networks were used to provide null expectations for the extent and nature of PPI network evolution. We found strong evidence for conservation of PPIs, with lower than expected levels of change in PPIs for about a quarter of the proteome. Furthermore, we found that changes in predicted PPI networks are poorly predicted by sequence divergence. Our analyses identified a number of functional classes experiencing fewer PPI changes than expected, suggestive of purifying selection on PPIs. Our results demonstrate the added benefit of considering predicted PPI networks when studying the evolution of closely related organisms.</p></div

Enriched GO terms for proteins with higher than expected γ not identified when analyzing positively selected protein sequences.

<p>Enriched GO terms for proteins with higher than expected γ not identified when analyzing positively selected protein sequences.</p

Enriched GO terms for proteins with lower than expected γ not identified when analyzing slowly evolving protein sequences.

<p>Enriched GO terms for proteins with lower than expected γ not identified when analyzing slowly evolving protein sequences.</p