Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport

Abstract

Key points: Small transmembrane proteins such as FXYDs, which interact with Na⁺ ,K⁺ -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca²⁺ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport.

Abstract: Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na⁺ ,K⁺ -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca²⁺ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

Keywords: FXYD proteins; Na+−K+−ATPase; SERCA; micropeptides; phospholemman; protein phosphorylation; vertebrate evolution.

Figure 1. Schematic overview of the coding sequence for FXYD homologue in Petromyzon marinus

A, grey boxes represent the predicted 4 exons of the proto‐FXYD5 in P. marinus. Hatched boxes below the exons indicate the qPCR‐primer binding sites that span the exon 1 to exon 2 (forward primer) and exon 3 to exon 4 (reverse primer) junction. The sequence between the black arrows are the results of the qPCR‐product Sanger sequencing and reveal the genomic cDNA sequence that encodes the LTYD protein motif (marked as black boxes above exons 2 and 3). The dotted line within the LTYD coding sequence indicates the unknown sequence of intron 2 that was spliced from the cDNA. B, the predicted coding sequence of proto‐FXYD5 including the predicted amino acid translation.

Figure 2. Tissue distribution of FXYD homologue in P. marinus

Expression of FXYD homologue (LTYD) mRNA was measured in different tissues by real‐time PCR. 18S rRNA was used as the endogenous control. A, C _t values for LTYD and 18S rRNA. B, relative expression levels of LTYD. Gene expression was normalized to the expression of 18S rRNA and expressed as fold change relative to the expression in brain using the ΔΔC _t method. C, agarose gel electrophoresis of real‐time PCR products from gills, liver, gut and heart.

Figure 3. Gene neighbourhoods of FXYDs

Neighbourhoods are presented schematically (i.e. not to scale). Black boxes denote FXYD genes and grey boxes denote conserved flanking genes. Pseudogenes are denoted by boxes with vertical lines and non‐coding RNAs by boxes with dots.

Figure 4. Gene neighbourhoods of phospholamban, sarcolipin and myoregulin

Neighbourhoods are presented schematically (i.e. not to scale). Black boxes denote genes for phospholamban (PLN), sarcolipin (SLN), or myoregulin (MRLN), while grey boxes denote conserved flanking genes. Pseudogenes are denoted by boxes with vertical lines and non‐coding RNAs by boxes with dots. Two phospholamban genes are shown for Astyanax mexicanus, a representative of bony fishes.

Figure 5. Neighbour‐joining tree of 13 FXYD gene subfamilies

Figure 6. Phosphorylation sites in subfamilies FXYD1, FXYD2, FXYD2f, FXYD3/4, FXYD3 and FXYD4

Phosphorylation sites, are graded on a scale from 0 (the least reliable) to 9 (the most reliable). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Phosphorylation sites in subfamilies FXYD5, FXYD6, FXYD6f, FXYD7 and FXYD8

Phosphorylation sites, are graded on a scale from 0 (the least reliable) to 9 (the most reliable). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8. Major events in the evolutionary history of small transmembrane regulators of ion transport

Figure 9. Schematic representation of the evolutionary history of FXYD genes

See the text for explanation of the evolutionary history of FXYDs.