Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock

Abstract

Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day-specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.

Figure 1. Affinity-tagged FRQ maintains physiological and molecular rhythms and reveals FRQ interactome and phosphorylation

(A) Race tube assay of wild-type (WT) and the frq^V5H6 strain (τ = period in hours, σ = standard deviation, n = number of race tubes). (B) Western blot of frq^V5H6 showing circadian rhythmic abundance and phosphorylation of FRQ over 48h detected with α-V5. Coomassie stained membranes serve as loading controls. (C) FRQ interactome identified by MS/MS after purification for 2 biological replicates. Listed is the number of unique peptides sequenced and their combined percent (%) mass; see Table S1. (D) Schematic of FRQ showing the position of translational start sites, domain structure, position and period length of previously characterized alleles, and newly identified in vivo phosphorylation sites. Regions of FRQ in green were either directly sequenced by MS/MS or do not contain S/T. Seventy-five S/T residues were unambiguously identified as phosphorylated (blue lines) and positions with low confidence are labeled ambiguous (purple lines). Sites marked by a red asterisk (*) are conserved over 120 million years of evolution. CC – coiled-coiled domain, NLS – nuclear localization signal, FCD – FRQ-CKI interacting domain, FFD – FRQ-FRH interacting domain, PEST-# - pest domains, frq^# - frq alleles followed by period length.

Figure 2. Dynamic changes in the FRQ-interactome provide a temporal framework for regulation of the WCC

(A) Schematic of the pooled reference sample design for the complete circadian day SILAC experiment (see text). Proteins were purified via FRQ and split to separately follow the interactome and FRQ phosphorylation. (B) Top – Representative MS spectra for doubly charged heavy and light FRQ peptides (387-DNGSASNSGGDQTELGGTGTGSGDGSGSGGR-417) show an observed m/z difference of 5.01 (6-¹³C and 4-¹⁵N in Arg) and a H/L ratio of ∼2. Bottom – Distribution of log₂ (H/L) for all sequenced FRQ and a sample of the 1:1 mix peptide pairs at CT12 (n = number of H/L ratios measured, μ = mean log₂ (H/L), σ = SD). (C) The FFC interacts with the WCC primarily in the early circadian day, phase-leading the interaction with CK1. Left – Mean log₂ (H/L) of all peptides sequenced for FRQ (diamond at CT12 = μ^FRQ-μ^1:1 from B), FRH, WC-1, WC-2, and CK1 at each time (± SD). Right – Same data in left panel normalized to FRQ. That levels of FRH closely track FRQ is seen by the near flat FRH line. Note: CT, circadian time, is calculated here sensu stricto, but period and phase differences arising from use of the SILAC strain means that the circadian biochemistry seen at CT4 (DD16) more closely matches that typically seen in the late subjective night in WT cultures (Figure S2).

Figure 3. Topological and phase specific changes in FRQ phosphorylation

(A) Dual molecular rhythms in FRQ abundance and phosphorylation. Top – Graph showing change in total (i.e. Figure 3C) and phosphorylated FRQ (calculated by averaging all log₂ (H/L) phosphopeptides). Early in the subjective day FRQ abundance and phosphorylation increase, while later phosphorylation plateaus as FRQ levels start to decline. Bottom – Heat map of individual FRQ phosphopeptides (rows) throughout the circadian day (CT in columns). (B) FRQ undergoes phase-specific phosphorylation in discrete domains. Left – Schematic of FRQ showing the position of phosphorylated residues identified in the full time course experiment. Right – Heat map representing the relative change in abundance of 63 phosphopeptides normalized to the average level of FRQ phosphorylation. Values tracking the mean appear black, while hyper- and hypophosphorylated residues are yellow and blue respectively. Numbers on the right of the heat map indicate the specific FRQ residue(s) phosphorylated for both (A) and (B).

Figure 4. Phosphorylation of FRQ leads to opposing effects on circadian rhythms and protein half-life

(A) A frq knock-in cassette (frq^KI) can restore rhythmicity to Δfrq. (B) Ablation of key phosphorylation sites in the PEST-1 domain results in lengthened periods. Representative luciferase traces show site-specific increases in period length of mutants. (C) Similar to (B) except these strains carry mutations in the early-phase phosphorylated C-terminal region of FRQ and result in decreased periods. (D) Summary of race tube (rt) and luciferase (luc) data showing the change (Δ) in period compared to a WT transformed control for the indicated phosphorylation site mutants. (E) Left – Representative Western blots of FRQ degradation after an LD transition for frq^S900A, frq^S548A, and frq^KI probed with α-V5. Right – Densitometric analysis of (E) showing exponential fit used for FRQ half-life calculation. (F) Plot of FRQ half-life vs. period length. FRQ half-life correlates with period length where shorter half-lives result in shorter periods. The black line represents the predicted correlation at 25°C based on Ruoff et al. (2005). Error bars cover the range of two independent estimates of FRQ half-life and the standard deviation for period.

Figure 5. Multi-site phosphorylation of PEST-1 is necessary for regulated FRQ turnover

(A) Amino acid sequence of PEST-1 domain indicating position of 5 in vivo phosphorylation sites in red. (B) Race tube analysis of strains bearing various PEST-1 mutations. The phosphomimetic mutation (S548D) maintains a WT period; while strains bearing multiple proximal mutations in the PEST-1 domain, with all sites in (A) changed to either Ala (frq^5XS→A) or Asp (frq^5XS→D), abolish overt circadian rhythms. (C) Luciferase assays of strains from (B) show a low amplitude molecular rhythm in frq^5XS→A. (D) Representative Western blot of frq^KI and PEST-1 mutants grown in constant light (LL) or after transfer to the dark for 8h (DD8). The arrow indicates slower migrating forms of FRQ that are absent from frq^5XS→A. Bottom - Densitometric analysis for 3 biological replicates (± SEM).

Figure 6. The C-terminus of FRQ is physically required for wild-type period

(A) Schematic of FRQ showing deleted region in frq^ΔC-term. This area encompasses all FRQ-stabilizing phosphorylation sites. (B) Western blot shows stable expression of FRQ^ΔC-term in LL. Arrowhead indicates hypophosphorylated FRQ. (C) Representative luciferase traces showing short a molecular rhythm in frq^ΔC-term (n= number of individual measurements). (D) Mutation of distal phosphorylation sites on FRQ show an additive effect of phase-specific phosphorylation. Race tube analysis of frq^S900A and the multiple mutant frq^{S548A & S900A}.

Figure 7. Model of FRQ phosphorylation and interactome

Symbol size represents abundance of FRQ or relative steady-state interaction of FRH, WCC and CKI with FRQ. FRQ and FRH are always in a complex regardless of FRQ levels or phosphorylation. Slightly after subjective dawn, as frq mRNA levels reach a maximum, FRQ and CK1 interaction peaks, phase-lagging WCC interaction. In the subjective day as total FRQ levels increase, interaction with the WCC decreases. During this phase, the C-terminal region of FRQ is at its maximum relative phosphorylation state, helping to stabilize FRQ. During the subjective evening/night phosphorylation of the PEST-1 domain increases thereby destabilizing FRQ and interaction with the WCC is minimal. Approaching subjective dawn FRQ becomes fully phosphorylated, is degraded, and frq expression is reinitiated. New, hypophosphorylated, FRQ immediately associates with FRH and quickly increases its interaction level with the WCC to start a new cycle.