SummaryCircadian (approximately daily) rhythms are a pervasive property of mammalian cells, tissues, and behaviour, ensuring physiological and metabolic adaptation to solar time. Models of daily cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of ‘clock proteins’ PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are thus considered essential negative regulators of the oscillation; a function supported by behavioural arrhythmicity of CRY-deficient mice when kept under constant conditions. Challenging this interpretation, however, we find evidence for persistent circadian rhythms in mouse behaviour and cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have a shorter period than wild type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and determination of period by casein kinase 1δ/ε activity to be maintained. In the absence of CRY-mediated transcriptional feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational. Our findings suggest that PER proteins normally act as signalling hubs that transduce timing information to the nucleus, imparting daily rhythms upon the activity of transcriptional effectors.Highlights➢PER/CRY-mediated negative feedback is dispensable for mammalian circadian timekeeping➢Circadian variation in PER2 levels persists in the absence of rhythmic Per2 transcription➢CK1 and GSK3 are plausible mechanistic components of a ‘cytoscillator’ mechanism➢CRY-mediated feedback repression imparts robustness to biological timekeepingIn briefCircadian turnover of mammalian clock protein PERIOD2 persists in the absence of canonical transcriptional feedback repression and rhythmic clock gene activity, demanding a re-evaluation of cellular clock function and evolution.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ϵ activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

Between 6-20% of the cellular proteome is under circadian control to tune cell function with cycles of environmental change. For cell viability, and to maintain volume within narrow limits, the osmotic pressure exerted by changes in the soluble proteome must be compensated. The mechanisms and consequences underlying compensation are not known. Here, we show in cultured mammalian cells and in vivo that compensation requires electroneutral active transport of Na+, K+, and Cl− through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes alter their electrical activity at different times of the day. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

Between 6–20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl− through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K + abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRYdeficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

13Circadian timekeeping in mammalian cells involves daily cycles of CRYPTOCHROME-dependent 14 transcriptional feedback repression. Ablation of CRY in mice leads to reduced growth and numerous 15 other phenotypes for reasons that are not well understood. Here, we find that cells adapt to CRY 16 deficiency by extensive remodelling of the proteome, phosphoproteome and ionome, with twice the 17 number of circadian-regulated proteins and phosphopeptides as well as increased rhythmic ion 18 transport compared to wild-type cells. CRY-deficient cells also have increased protein synthesis and 19 reduced proteasomal degradation, as well as an altered energetic state. These adaptations render cells 20 more sensitive to stress, and may provide an explanation for the wide-ranging phenotypes of CRY-21 deficient mice. We suggest that daily rhythms in cellular protein abundance are damped by CRY-22 mediated repression to facilitate daily cycles of proteome renewal whilst maintaining protein 23 homeostasis. 24 25 complex containing the activating transcription factors (BMAL1 and CLOCK or NPAS2). The 52 stability, interactions and nucleocytoplasmic shuttling of the encoded PER and CRY proteins is 53 regulated post-translationally until, many hours later, they repress the activity of BMAL1-containing 54 complexes. This transcriptional-translational feedback loop (TTFL) is proposed as the basis of 55 circadian timekeeping in mammalian cells [15]. Genes whose transcription are regulated by core 56 TTFL factors are thought to drive circadian rhythms in the encoded proteins to control myriad cellular 57 functions [13,14]. 58 59 Within the TTFL model of circadian rhythm generation, CRY proteins are the essential repressors of 60 CLOCK/BMAL1 activity [16,17], and have long been considered indispensable for circadian 61 rhythms in vivo and cells ex vivo [18][19][20][21][22]. In contrast, PER proteins play critical signalling and 62 scaffolding roles, required for the nuclear import and targeting of CRY to BMAL1-containing 63 complexes [17]. Recently however, we found that CRY-deficient cells, tissues and mice retain the 64 capacity for circadian timing, in the absence of canonical TTFL function [23]. Similarly, circadian 65 oscillations were retained in cells and tissue slices lacking BMAL1 [24]. Whilst we cannot exclude 66 the presence of some unknown transcriptional feedback-driven oscillation, many previous 67 observations argue against this [12,17,19,21,22,[25][26][27][28]. Recent evidence supports the hypothesis that 68 a conserved post-translational cytosolic oscillator (PTO, or "cytoscillator") may be responsible for 69 generating the oscillation from which circadian transcriptional cycles derive [12,29,30]. Thus, while 70 the TTFL is crucial for rhythmic robustness and co-ordinating outputs, it is not required for generation 71 of rhythms. 72 73The complex phenotype of mice lacking CRY proteins has been interpreted to mean that circadian 74 timekeeping is crucial for organismal physiology. The emerging observations that circadia...

Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different protein labelling strategies, we show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results indaily consolidationof proteome renewal whilst minimising changes in composition. By combining mass spectrometry with pulsedisotopic labelling of nascently synthesised proteins, we gain direct insight into the relationship between protein synthesis and abundance proteome-wide, revealing that coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, such as ribosomes, RNA polymerase, and chaperonin complex. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythmsin the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of turnover.

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