A Magnesium Transport Protein Related to Mammalian SLC41 and Bacterial MgtE Contributes to Circadian Timekeeping in a Unicellular Green Alga

Abstract

Circadian clocks in eukaryotes involve both transcriptional-translational feedback loops, post-translational regulation, and metabolic, non-transcriptional oscillations. We recently identified the involvement of circadian oscillations in the intracellular concentrations of magnesium ions (Mg²⁺_i) that were conserved in three eukaryotic kingdoms. Mg²⁺_i in turn contributes to transcriptional clock properties of period and amplitude, and can function as a zeitgeber to define phase. However, the mechanism-or mechanisms-responsible for the generation of Mg²⁺_i oscillations, and whether these are functionally conserved across taxonomic groups, remain elusive. We employed the cellular clock model Ostreococcustauri to provide a first study of an MgtE domain-containing protein in the green lineage. OtMgtE shares homology with the mammalian SLC41A1 magnesium/sodium antiporter, which has previously been implicated in maintaining clock period. Using genetic overexpression, we found that OtMgtE contributes to both timekeeping and daily changes in Mg²⁺_i. However, pharmacological experiments and protein sequence analyses indicated that critical differences exist between OtMgtE and either the ancestral MgtE channel or the mammalian SLC41 antiporters. We concluded that even though MgtE domain-containing proteins are only distantly related, these proteins retain a shared role in contributing to cellular timekeeping and the regulation of Mg²⁺_i.

Keywords: Ostreococcus tauri; cellular rhythms; circadian clocks; magnesium transport; transporter proteins.

Figure 1

Overexpression of OtMgtE induces a long period phenotype. (a) CCA1 bioluminescent traces of OtMgtE-OX (blue) compared to the parent line (black) in free running conditions (constant light). The graph shows a line through discrete time points, at a ~1-h sampling rate (mean ± Standard Error Mean (SEM)). (b) Free-running period inferred from traces in (a), student’s t-test, **** p < 0.0001. (c) Relative OtMgtE messenger RNA (mRNA) levels in the parent line compared to OtMgtE-OX at ZT0 (dawn) and ZT12 (dusk). * = p < 0.05; *** = p < 0.001; student’s t-test. (d) Diurnal transcriptional expression profile of OtMgtE based on publicly available microarray data [31].

Figure 2

Overexpression of OtMgtE increases [Mg²⁺]_i at ZT0. Quantification of [Mg²⁺]_i by inductively coupled plasma mass spectrometry (ICP-MS) (a) or luciferase-based plate assays (b). A significant increase in [Mg²⁺]_i at dusk compared to dawn is observed in the parent line, and OtMgtE overexpression leads to an increase in [Mg²⁺]_i at ZT0. Non-significant (ns) = p > 0.05; * = p < 0.05; ** = p < 0.001; student’s t-test.

Figure 3

Overexpression of OtMgtE affects circadian period synergistically with cobalt(III)hexamine (CHA). Luminescent traces of the parent line (a) and OtMgtE-OX (b) at certain CHA concentrations in constant light. The graph shows a line through discrete time points, at a ~1-h sampling rate (mean ± SEM). (c) The dose-response curve of circadian period for the parent and the OtMgtE overexpressing line at increasing concentrations of CHA. ns: p > 0.05 ***: p < 0.005; student’s t-test.

Figure 4

Overexpression of OtMgtE does not rescue the effect of low Mg²⁺ on the circadian period. Luminescent traces of (a) the parent line and (b) OtMgtE-OX at different concentrations of extracellular Mg²⁺. The graph shows a line through discrete time points, at a ~1-h sampling rate (mean ± SEM). (c) Free-running period calculated from bioluminescent traces in (a,b). ns: p > 0.05 **: p < 0.01 ****: p < 0.0001; student’s t test.

Figure 5

Phylogenetic comparison of OtMgtE with eukaryotic and prokaryotic homologues. (a) Summary table of the distribution of proteins containing MgtE domains in eukaryotes and domain structure; we identified MgtE domains in species labelled in black but not those in grey. (b) Maximum-likelihood phylogenetic tree (based on 100 bootstraps) of MgtE domains in selected species. (x) denotes different homologue numbers from the same species, *x denotes the domain copy number within a homologue.

Figure 5

Phylogenetic comparison of OtMgtE with eukaryotic and prokaryotic homologues. (a) Summary table of the distribution of proteins containing MgtE domains in eukaryotes and domain structure; we identified MgtE domains in species labelled in black but not those in grey. (b) Maximum-likelihood phylogenetic tree (based on 100 bootstraps) of MgtE domains in selected species. (x) denotes different homologue numbers from the same species, *x denotes the domain copy number within a homologue.

Figure 6

Effect of known inhibitors of SLC41 on CCA1 expression in Ostreococcus tauri. Luminescent traces of the CCA1 bioluminescent line (parent line) at a range of concentrations of (a) amiloride or (b) imipramine. The graph shows a line through discrete time points at a ~1-h sampling rate (mean ± SEM). LL: Constant light.