Abstract
Circadian clocks have arisen in most eukaryotes and some prokaryotes to anticipate the rhythmic environmental changes imposed by Earth’s daily rotation on itself. These daily timekeeping mechanisms provide a competitive advantage for species as they allow for the appropriate allocation of daily cellular energy. Over the past few years, circadian research has increasingly focussed on identifying clock components responsible for rhythmic metabolism that are highly conserved across distinct taxonomic groups. Recent work identified circadian rhythms in the intracellular concentrations of Mg²⁺ ([Mg²⁺]i) in species representative of three eukaryotic kingdoms (plants, fungi, and animals). [Mg²⁺]i rhythms were identified as a core clock component, as alteration in [Mg2+]i had an effect on period, phase, and amplitude of the clock, conserved across species whose last common ancestor, LECA, existed more than a billion years ago. How [Mg2+]i rhythms are generated, and if they are generated from conserved mechanisms across eukaryotes, is currently unknown. Dynamic changes in [Mg2+]i are facilitated by membrane-bound transport proteins, either by passive transport (channels) or active transport (for example exchangers) and such proteins can be located on the plasma or organellar membranes. Mg²⁺ transport remains understudied in comparison to other types of ion transport, and the characterisation of Mg²⁺ transport proteins remains largely limited to prokaryotes, animals, plants, and fungi. We reviewed all characterised Mg²⁺ transporting proteins across eukaryotic species and explored, in silico, the extent of protein conservation in proteomes representative of the main eukaryotic super-groups. We found that protein families with prokaryotic members had the highest degree of conservation across eukaryotes, hypothesising that proteins lacking prokaryotic ancestors have emerged independently across taxa. Furthermore, we suggest that proteins with pre-eukaryotic homologs are likely to be the proteins first involved in generating [Mg²⁺]i in LECA, highlighting their evolutionary importance. The algal clock model Ostreococcus tauri has six proteins with pre-eukaryotic homologs: OtMgtE, OtMRS2.1/2/3, and OtCNMM1/2. We characterised two of these proteins, OtMgtE and OtCNNM2, showing that both proteins contribute to timekeeping. Gene overexpression for both OtMgtE and OtCNNM2 generated a long-period phenotype, and was accompanied by changes in [Mg²⁺]i. OtMgtE overexpression increased [Mg²⁺]i at dawn and OtCNNM2 overexpression decreased [Mg²⁺]i both at dawn and at dusk. Rhythmic [Mg²⁺]i implies that cellular pathways have rhythmic access to available Mg²⁺, which could fine-tune the activity of relevant proteins over the 24 hours of the day. To investigate which proteins (and associated pathways) are sensitive to changes in [Mg²⁺]i we generated Ostreococcus cultures acclimated to low [Mg²⁺]e. Ostreococcus cells acclimated to low [Mg²⁺]e displayed a similar growth rate to control cells grown at normal [Mg²⁺]e, but did so with a significant decrease in [Mg²⁺]i. Mass spectrometry identified the proteins that were significantly differentially regulated to maintain cellular homeostasis despite the change in Mg²⁺ content of the cell. We found that the abundance of proteins involved in metabolic pathways such as protein synthesis, folding, and degradation, and ATP generation were altered, providing a list of proteins the activity of which could be rhythmically regulated by Mg²⁺ under physiological conditions. Furthermore, as changes in the abundance of proteins are required to maintain cellular homeostasis in acclimated cells, we also hypothesise that dynamic cellular Mg²⁺ use might in itself play a role in contributing to rhythmic [Mg²⁺]i by changing the state of the ion from bound to free form. This thesis provides a first insight into the Ostreococcus Mg²⁺ transport system, and its circadian regulation. Additional Ostreococcus Mg²⁺ transporters need to be characterised, as well as their relationship with the cellular pathways that rhythmically use Mg²⁺, to fully comprehend the spatiotemporal parameters of daily Mg²⁺ biology in eukaryotic cells.
