A PEST signal reduces luciferase stability.

<p>(<b>A</b>) Schematic representation of the luciferase reporter expressed under control of the promoter of the <i>frequency</i> gene (<i>frq promoter</i>). The coding region of the firefly luciferase has been codon optimized for <i>Neurospora crassa</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083660#pone.0083660-Gooch1" target="_blank">[19]</a>. The PEST sequence fused to the 3′-end of the luciferase is indicated. The reporter gene was integrated into the genome downstream of the his-3 locus. (<b>B</b>) A protein stability assay in light grown cells was carried out in the presence of cycloheximide (CHX) to compare the degradation of LUC-PEST and unmodified LUC. Mycelia were grown in liquid culture at 25°C and CHX was added at t = 0 at a final concentration of 10 µg/ml. Cells were harvested at the indicated time points, native protein extracts were prepared and luciferase activity was analyzed as described in Material and Methods. (Left) Absolute bioluminescence levels at t = 0 are shown. (Right) Bioluminescence levels are blotted over time. Levels at t = 0 were set to 100% (Error bars indicate to ± SEM, <i>n</i> = 3). Exponential trend lines are fitted to the data (values <5% were excluded). (<b>C</b>) Cultures of <i>frq::luc</i> (blue line) and <i>frq::luc-PEST</i> (orange line), grown in darkness under growth-restricting conditions on solid medium in a 96-well plate, were exposed to a 1 min light pulse and then released into constant darkness. Luciferase activity was recorded <i>in vivo</i> for 5 hours in 20 min intervals. <i>frq</i> mRNA levels (black line) were determined by qPCR in <i>WT</i> strain grown in liquid medium. Data were normalized by setting the maxima to 100%. (Error bars indicate ± SEM, <i>n</i> = 3).