Figure 1.
Components of the muskelin/RanBPM/CTLH complex.
A, Scaled schematic diagrams of the domain organisation of muskelin/RanBPM/CTLH and GID complex proteins. Muskelin is not encoded in budding yeast. Shared domains are identified in the key. B, Muskelin/RanBPM/CTLH complex components are conserved across the domain of eukaryotes. The taxonomic tree of representative eukaryotic species is rendered in TreeDyn. Asterisks indicate the encoding of MRCTLH complex proteins in the indicated species. Absence of an MRCTLH complex proteins reflects absence of orthologues in other species of the same taxonomic lineage.
Table 1.
Plant species with multiple genes encoding MRCTLH proteins.
Figure 2.
Phylogenetic relationships of RanBPM, TWA1, MAEA and Rmnd5, based on their LisH/CTLH regions.
109 sequences spanning the LisH and CTLH domains of these proteins from species representing all the eukaryotic lineages in which each of these MRCTLH protein is distributed, but excluding parasitic species (100 aa; species orthologues in the dataset are identified in Figure S4) were aligned in Sate via MAFFT and MUSCLE. Phylogenetic trees were constructed in PhyML (A) or Mr. Bayes (B) and are presented as unrooted trees with proportionate branch lengths. In A and B, the clade for each protein is labelled. Scale bars indicate substitutions/site. In A, branch support values are bootstrap values from 1000 cycles/SH ratios; in B, figures are posterior probability values.
Figure 3.
Analysis of the LisH domains of RanBPM, TWA1, MAEA and Rmnd5 by Sequence Logos.
A, B, structure of the LisH domain of Lis1. Structural diagrams show side (A) and top (B) views of the two LisH domains that form an antiparallel dimer in human Lis1 (PDB 1UUJ). C, Sequence Logo consensus of the LisH domain, derived from all LisH domains in the SMART database. Amino acids are in single letter code with basic residues in blue, acidic residues in red, hydrophobic residues in black, polar residues in green, and amide-containing residues in pink. Asterisks indicate the most highly-conserved residues. The adjacent side views of a single LisH domain from Lis1 (lefthand, showing inner face, righthand showing outer face according to the dimer structure) identify the positions of these residues in colours corresponding to the Logo. D–G, Sequence Logos for the LisH domain of the indicated MRCTLH complex proteins. For each Logo, the positions of positively charged residues (blue) and negatively charged residues (red) with bit scores ≥2 are mapped onto the structure of a single LisH domain from Lis1 (lefthand shows inner face, righthand shows outer face according to the dimer structure).
Figure 4.
Analysis of the CTLH domains of RanBPM, TWA1, MAEA and Rmnd5 proteins by Sequence Logos.
A, The Sequence Logo consensus for CTLH domains, derived from all CTLH domains in the SMART database. B–F, Sequence Logos corresponding to the CTLH domain of the indicated MRCTLH complex proteins. Colour coding as in Fig. 3.
Figure 5.
Comparison of subcellular localisations of RanBPM, TWA1, MAEA and Rmnd5.
Localisations of epitope-tagged RanBP9, TWA1, Rmnd5a or MAEA and their N- and C-terminal deletion proteins (in the schematics, L = LisH domain, H = CTLH domain) were visualised in fixed COS-7 cells by immunofluorescence and laser scanning confocal microscopy. Images are in the XY plane unless otherwise stated. The merged images show each protein (in green or red) and DAPI-stained nuclei (in blue). Insets of the boxed areas show, for clarity, the corresponding single channel images for RanBP9 C-terminal protein, full-length Rmnd5a, or Rmnd5a N-terminal protein. Arrow in the image of Rmnd5a indicates small nuclear particles; arrow in the image of MAEA N-terminal protein indicates small cytoplasmic particles; arrowhead in image of MAEA C-terminal protein indicates cytoplasmic granules. Cells shown are representative of three independent experiments. Bars = 25 µm.
Figure 6.
Analysis of the RING domains of Rmnd5 and MAEA.
A, TRIM32 RING domain structure (PDB 2CT2). The 8 zinc-coordinating residues are shaded green or purple to distinguish between the zinc-coordinating sites S1 or S2, respectively. S1 and S2 are linked by two residues (yellow). Strongly conserved proline residues adjacent to cysteine residues of the RING domains are marked with a blue asterisk. B, consensus sequence Logo of RING domains from the SMART database. Green or purple arrows indicate the zinc-coordinating residues of sites S1 or S2, respectively. Yellow asterisks indicate residues linking S1 and S2. C, D, Sequence Logos of RING domains from Rmnd5 [C] or MAEA (D) orthologues. In all Logos, residues corresponding to the 8 zinc-coordinating residues are numbered as in 6B. Blue asterisks in C and D indicate strongly conserved prolines at the equivalent position to the well-conserved proline of the TRIM32 RING domain. The three residue gap in the Rmnd5 RING Logo is due to the extended sequence of C. briggsae Rmnd5 RING domain.
Figure 7.
Model for the evolution of the MRCTLH complex.
The model proposes that all proteins except muskelin were present in the last eukaryotic common ancestor, also that RanBPM and TWA1 vs MAEA and Rmnd5 share common ancestors, respectively. Rm = Rmnd5; Ma = MAEA; T = TWA1; RB = RanBPM. See Discussion for details.