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.