Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

Abstract

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

Genome-wide identification of resistance genes and transcriptome regulation in yeast to accommodate ammonium toxicity

“…In contrast, gtr1Δ reduces the activity of TORC1, which increases the expression level of nitrogen transporters and ammonium consumption but reduces amino acid consumption [ 36 ]. GTR2 encodes a Ras-like small GTPase that plays a role in regulating nutrition-responsive TORC1 kinase signal transduction, exocytosis sorting of endosomes, and epigenetic control of gene expression [ 37 ]. Deletion of GTR1 or GTR2 may render cells incapable of activating TORC1, resulting in cell toxicity due to the inability to degrade ammonia in vivo under high ammonia stress.…”

The Case for the Target of Rapamycin Pathway as a Candidate Circadian Oscillator

“…The gene product (which we named VTA, vacuolar TOR-associated protein) was identified as a component of the TOR (Target of Rapamycin) pathway [27] and is homologous to a component of the EGO complex in yeast and Ragulator complex in mammals (see below). We have also studied a binding partner of VTA, one of the Rag proteins homologous to Gtr2 in yeast (see below) and have shown that a knockout of gtr2 in Neurospora has a phenotype very similar to the vta knockout, abolishing FRQ-less rhythmicity [29]. These results have focussed our lab's attention on the connections between the TOR pathway and circadian rhythmicity and raise the possibility that TOR is involved in the generation of FRQ-less oscillations in Neurospora.…”

“…In our lab, we have found that knockouts of two TOR pathway components in Neurospora, vta and gtr2, dampen the rhythm of conidiation and also damp out the FRQ protein rhythm [28,29], indicating that these gene products are required to maintain rhythmicity of FRQ protein. When taken together with the findings of Diernfellner et al [65], this may indicate that TOR activity contributes to the rhythmicity of FRQ protein phosphorylation and stability.…”

“…Neurospora has provided evidence for a role for TOR pathway components in sustaining FRQ-less conidiation rhythms. The free-running rhythm of conidiation seen in FRQ-less strains under low-choline conditions can be abolished using knockouts of the two TOR pathway components vta and gtr2, and the entrained rhythm of conidiation under choline-sufficient conditions in FRQ-less strains is also severely impacted by these gene knockouts [28,29].…”

Studying the Human Microbiota: Advances in Understanding the Fundamentals, Origin, and Evolution of Biological Timekeeping

“…Collectively, these could potentially function as quasi-universal metabolic oscillators across eukaryotic organisms. However, considering that red blood cells do not possess the TOR pathway, yet their central metabolic pathways demonstrate robust rhythmicity, various metabolic oscillators may exist inside cells [400][401][402].…”