Abstract
Ubiquitination is an essential post-translational modification that regulates signalling and protein turnover in eukaryotic cells. Specificity of ubiquitination is driven by ubiquitin E3 ligases, many of which remain poorly understood. One such is the mammalian muskelin/RanBP9/CTLH complex that includes eight proteins, five of which (RanBP9/RanBPM, TWA1, MAEA, Rmnd5 and muskelin), share striking similarities of domain architecture and have been implicated in regulation of cell organisation. In budding yeast, the homologous GID complex acts to down-regulate gluconeogenesis. In both complexes, Rmnd5/GID2 corresponds to a RING ubiquitin ligase. To better understand this E3 ligase system, we conducted molecular phylogenetic and sequence analyses of the related components. TWA1, Rmnd5, MAEA and WDR26 are conserved throughout all eukaryotic supergroups, albeit WDR26 was not identified in Rhizaria. RanBPM is absent from Excavates and from some sub-lineages. Armc8 and c17orf39 were represented across unikonts but in bikonts were identified only in Viridiplantae and in O. trifallax within alveolates. Muskelin is present only in Opisthokonts. Phylogenetic and sequence analyses of the shared LisH and CTLH domains of RanBPM, TWA1, MAEA and Rmnd5 revealed closer relationships and profiles of conserved residues between, respectively, Rmnd5 and MAEA, and RanBPM and TWA1. Rmnd5 and MAEA are also related by the presence of conserved, variant RING domains. Examination of how N- or C-terminal domain deletions alter the sub-cellular localisation of each protein in mammalian cells identified distinct contributions of the LisH domains to protein localisation or folding/stability. In conclusion, all components except muskelin are inferred to have been present in the last eukaryotic common ancestor. Diversification of this ligase complex in different eukaryotic lineages may result from the apparently fast evolution of RanBPM, differing requirements for WDR26, Armc8 or c17orf39, and the origin of muskelin in opisthokonts as a RanBPM-binding protein.
Highlights
Ubiquitin-dependent proteolysis of proteins by the proteasome is a major cellular mechanism for rapid modulation of protein levels in eukaryotic cells, that likely has common ancestry with a prototypic system identified in Archaea [1]
As a first step to understand the nature of the glucose induced degradation deficient (GID) and Muskelin/RanBP9/CTLH complex (MRCTLH) complexes in greater depth, we have examined the evolution of individual components through analyses of prokaryotic and eukaryotic genomes and phylogenetic inference
Starting from extensive sequence database searches, we have combined molecular, phylogenetic, and initial experimental analyses to better understand the molecular phylogeny of the proteins of the MRCTLH/GID complex
Summary
Ubiquitin-dependent proteolysis of proteins by the proteasome is a major cellular mechanism for rapid modulation of protein levels in eukaryotic cells, that likely has common ancestry with a prototypic system identified in Archaea [1]. The ability to degrade proteins efficiently and via the Ubiquitin Proteasome Pathway (UPP) is essential for many functions of eukaryotic cells. Ubiquitination occurs through a pathway catalyzed by ubiquitin-activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin-protein ligases (E3) [5]. Two E1 enzymes, over 35 E2 enzymes and over 600 E3 ubiquitin ligases are encoded in the human genome [4,5,6]. Many E3 enzymes are characterised by one of two protein domains that are essential for E3 ligase activity: the Homologous to the E6-AP Carboxyl (HECT) domain or the Really Interesting New Gene (RING) domain [4]. HECT E3s accept activated ubiquitin from the E2 before substrate modification, whereas RING E3s act as scaffolds that bind the substrate and the ubiquitin-loaded E2 concurrently. Additional variability is achieved by RING domain proteins that participate in multi-protein complexes, in which other proteins act as adaptors for recognition or stabilisation of interactions with the specific substrate [7,8]
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