Proteasome function is required for biological timing throughout the twenty-four hour cycle

Abstract

Circadian clocks were, until recently, seen as a consequence of rhythmic transcription of clock components, directed by transcriptional/translational feedback loops (TTFLs). Oscillations of protein modification were then discovered in cyanobacteria. Canonical posttranslational signaling processes have known importance for clocks across taxa. More recently, evidence from the unicellular eukaryote Ostreococcus tauri revealed a transcription-independent, rhythmic protein modification shared in anucleate human cells. In this study, the Ostreococcus system reveals a central role for targeted protein degradation in the mechanism of circadian timing. The Ostreococcus clockwork contains a TTFL involving the morning-expressed CCA1 and evening-expressed TOC1 proteins. Cellular CCA1 and TOC1 protein content and degradation rates are analyzed qualitatively and quantitatively using luciferase reporter fusion proteins. CCA1 protein degradation rates, measured in high time resolution, feature a sharp clock-regulated peak under constant conditions. TOC1 degradation peaks in response to darkness. Targeted protein degradation, unlike transcription and translation, is shown to be essential to sustain TTFL rhythmicity throughout the circadian cycle. Although proteasomal degradation is not necessary for sustained posttranslational oscillations in transcriptionally inactive cells, TTFL and posttranslational oscillators are normally coupled, and proteasome function is crucial to sustain both.

Figure 1

CCA1-LUC and TOC1-LUC Degradation Rates under Different Light Regimes (A) Degradation rates of CCA1-LUC (blue traces) and TOC1-LUC (red traces) calculated from the curve fitting to the exponential phase of decay following inhibition of de novo protein synthesis with cycloheximide. The x axis indicates treatment time; light regime is indicated in the panels. Error bars represent standard error of the mean (SEM; n = 5). Decay rates measured for free luciferase ranged from 0.165 to 0.136 hr⁻¹, as indicated by the horizontal dotted lines. (B) Number of CCA1-LUC (blue line) or TOC1-LUC (red line) molecules/cell for an LD12:12 cycle calculated by in vitro luciferase activity of cell extracts (mean values plotted ± SEM; n = 2). (C) Absolute degradation rates in molecules/cell/hr for CCA1-LUC (blue lines) and TOC1-LUC (red lines) obtained from multiplying decay rates by molecule number (mean values plotted ± SEM; n = 2). See also Figure S1.

Figure 2

Effects of Proteasomal Inhibition (A) Period difference relative to vehicle-treated cells, resulting from treatment with indicated concentrations of epoxomicin or PYR-41 on CCA1-LUC (red bars) or pCCA1::LUC (black bars). Error bars represent standard deviation (SD; n = 8). (B) Effect of epoxomicin (blue traces, n = 4) or MG132 (red traces, n = 8) on CCA1-LUC in downward (top) or upward (bottom) phase, compared to vehicle (black traces, n = 8). See also Figure S2.

Figure 3

Proteasomal Inhibition Stops TTFL Rhythmicity Phase-Independently (A) Application of saturating concentrations of MG132 (40 μM, red traces) or vehicle (black traces) to CCA1-LUC cells in constant light. (B) Examples from wedge data of peak phases of individual wells (n ≥ 6) of CCA1-LUC cells subjected to various (0–24) hours of proteasomal inhibition (red wedge, right) or vehicle (gray wedge, left) starting at ZT0 and ending by wash off. (C) Summary of phase shifts (error bars represent SD, n ≥ 6) relative to vehicle-treated controls for all treatment durations (x axis) and starting times. Two black lines represent the expected result, assuming either total resetting by wash off (hypothesis 1) or no effect (hypothesis 2). See also Figure S3.

Figure 4

Application of MG132 Arrests Cytosolic Oscillations (A) Three independent 48 hr time series of protein extracts in constant light with vehicle or MG132 treatment starting after 12 hr. Samples were run on immunoblots using a PRX-SO_2/3 antibody. For equal loading control, Coomassie staining of Rubisco (RbcL) is shown on a representative gel (bottom panels). (B) Densitometry performed with ImageJ64 showing grouped data of the three replicates for vehicle-treated (black line) or MG132-treated (blue line) cells. Dotted lines indicate SD; blue shaded area indicates window of inhibition. See also Figure S4.