<i>P</i>. <i>infestans</i> eEF3 possesses ribosome-stimulated ATPase activity.

<p>ATPase activity of <i>S</i>. <i>cerevisiae</i> (■) and <i>P</i>. <i>infestans</i> (●) eEF3 was assayed in the presence (filled) or absence (empty) of <i>S</i>. <i>cerevisiae</i> ribosomes. The P_i released was measured using the PiColorLock Gold Phosphate Detection System. Reactions included varying amounts of eEF3, 25 nM ribosomes and 1 mM ATP and were carried out at room temperature for 30 min.</p

<i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i> and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 complement the loss of <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i> eEF3.

<p>(A) Yeast strains expressing eEF3 from the indicated species as the only form of eEF3 were streaked onto YEPD medium and incubated at 30°C for 2 d. CEN–low copy number plasmid/low expression. 2μ –high copy number plasmid/high expression. (B) Growth curves were generated from A₆₀₀ measurements of exponentially growing cultures over the indicated time period. Error bars represent standard error. (C) Whole cell extracts were prepared from the indicated strains, separated by SDS-PAGE, and stained with Ponceau S (bottom panel). The membrane was then cut and immunoblotted with either an anti-His antibody to detect eEF3 or anti-Pgk1 antibody as a loading control. The control lane is an extract from a yeast strain (TKY1617) that does not express epitope tagged eEF3.</p

<i>S</i>. <i>cerevisiae</i> strains used in this study.

<p><i>S</i>. <i>cerevisiae</i> strains used in this study.</p

Domain specific differences in the conservation of eEF3.

<p>(A) Individual domains of <i>S</i>.<i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 were aligned using Clustal Omega and the percentage identity to <i>S</i>. <i>cerevisiae</i> eEF3 is shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref023" target="_blank">23</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref024" target="_blank">24</a>]. The amino acids comprising each domain in <i>S</i>. <i>cerevisiae</i> are indicated. (B) Alignment of the chromodomain of <i>S</i>. <i>cerevisiae</i>, <i>S</i>. <i>pombe</i>, and <i>P</i>. <i>infestans</i> eEF3 was performed using Clustal Omega and shading based on identity was done using Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190524#pone.0190524.ref025" target="_blank">25</a>]. Dark gray represents identity in all three species and light gray represents identity in two species. (C) Maximum likelihood tree with 500 bootstrap replicates created using phylogeny.fr.</p

<i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i> and <i>P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> eEF3 complement the loss of <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i> eEF3.

<p>(A) Yeast strains expressing eEF3 from the indicated species as the only form of eEF3 were streaked onto YEPD medium and incubated at 30°C for 2 d. CEN–low copy number plasmid/low expression. 2μ –high copy number plasmid/high expression. (B) Growth curves were generated from A₆₀₀ measurements of exponentially growing cultures over the indicated time period. Error bars represent standard error. (C) Whole cell extracts were prepared from the indicated strains, separated by SDS-PAGE, and stained with Ponceau S (bottom panel). The membrane was then cut and immunoblotted with either an anti-His antibody to detect eEF3 or anti-Pgk1 antibody as a loading control. The control lane is an extract from a yeast strain (TKY1617) that does not express epitope tagged eEF3.</p

<i>P</i>. <i>infestans</i> eEF3 possesses ribosome-stimulated ATPase activity.

<p>ATPase activity of <i>S</i>. <i>cerevisiae</i> (■) and <i>P</i>. <i>infestans</i> (●) eEF3 was assayed in the presence (filled) or absence (empty) of <i>S</i>. <i>cerevisiae</i> ribosomes. The P_i released was measured using the PiColorLock Gold Phosphate Detection System. Reactions included varying amounts of eEF3, 25 nM ribosomes and 1 mM ATP and were carried out at room temperature for 30 min.</p

Strains expressing <i>P</i>. <i>infestans</i> eEF3 show defects in translation elongation and/or termination.

<p>(A) Antibiotic sensitivity was determined by measuring the diameter of the zone of growth inhibition around a disk containing 10 μL of the indicated drug: Paromomycin (800 mg/ml), hygromycin (25 mM), and cycloheximide (1 mM). The graph represents the average of three experiments and error bars representing the standard error are shown. (B) Ribosome extracts were prepared from the indicated strains in the absence of cycloheximide. Extracts were analyzed by 7–47% sucrose density gradient centrifugation and representative A₂₅₄ traces are shown. The area under the 80S and polyribosome peaks was analyzed using Image J and the ratio of polyribosomes to monosomes (p/m) is indicated for each strain. <i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, <i>and P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i>.</p

Strains expressing <i>P</i>. <i>infestans</i> eEF3 exhibit a reduction in protein synthesis.

<p><i>S</i>. <i>cerevisiae</i> strains expressing eEF3 from the indicated species (<i>S</i>. <i>cerevisiae (S</i>.<i>c</i>.<i>)</i>, <i>S</i>. <i>pombe (S</i>.<i>p</i>.<i>)</i>, <i>and P</i>. <i>infestans (P</i>.<i>i</i>.<i>)</i> were grown to log phase in C-MET at 30°C. [³⁵S] methionine was added and total protein synthesis was measured by tricholoroacetic acid precipitation. Incorporation (counts per min) is expressed per A₆₀₀ unit. Each time point was performed in triplicate and error bars represent standard error. A representative experiment is shown.</p