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).