The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers

Abstract

Background: The rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins. They are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.

Results: Phylogenetic tree analysis carried out using several independent methods for tree constructions and the corresponding statistical tests suggests that, despite its broad distribution in all three superkingdoms, the rhomboid family was not present in the last universal common ancestor of extant life forms. Instead, we propose that rhomboids evolved in bacteria and have been acquired by archaea and eukaryotes through several independent horizontal gene transfers. In eukaryotes, two distinct, ancient acquisitions apparently gave rise to the two major subfamilies, typified by rhomboid and PARL (presenilins-associated rhomboid-like protein), respectively. Subsequent evolution of the rhomboid family in eukaryotes proceeded by multiple duplications and functional diversification through the addition of extra transmembrane helices and other domains in different orientations relative to the conserved core that harbors the protease activity.

Conclusions: Although the near-universal presence of the rhomboid family in bacteria, archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor, phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer. This emphasizes the importance of explicit phylogenetic analysis for the reconstruction of ancestral life forms. A hypothetical scenario for the origin of intracellular membrane proteases from membrane transporters is proposed.

Figure 1

Multiple alignment of the conserved core of the rhomboid family proteins. The alignment includes the majority of the detected rhomboid family proteins; some closely related sequences were omitted. Only the six conserved (predicted) transmembrane helices (TMH) and short surrounding regions are shown. The boundaries of the predicted TMH are indicated by gray shading and overline and they are numbered 1-6. The number of amino-acid residues in the omitted terminal and internal regions are indicated. The consensus shows amino-acid residues present in at least 90% of the aligned sequences; h stands for hydrophobic residues (A, C, I, L, V, M, F, Y, W in the single-letter amino-acid code) and s for small residues (G, A, S, D, N, V). The proposed catalytic serine (TMH4) and histidine (TMH6) as well as conserved residues in TMH2 with possible ancillary roles in catalysis are highlighted in color. The proteins are identified with the gene identification (GI) number from the nonredundant database and an abbreviated species name. Bacterial species are color-coded green, eukaryotic species blue and archaeal species yellow. Species name abbreviations: Aerpe, Aeropyrum pernix; Agrtu, Agrobacterium tumefaciens; Anoga, Anopheles gambiae; Arath, Arabidopsis thaliana; Arcfu, Archaeoglobus fulgidus; Bacsu, Bacillus subtilis; Brume, Brucella melitensis; Caeel, Caenorhabditis elegans; Caucr, Caulobacter crescentus; Chlte, Chlorobium tepidum; Cloac, Clostridium acetobutilicum; Corgl, Corynebacterium glutamicum; Deira, Deinococcus radiodurans; Dicdi, Dictyostelium discoideum; Drome, Drosophila melanogaster; Escco, Escherichia coli; Haein, Haemophilus influenzae; Halsp, Halobacterium sp.; Homsa, Homo sapiens; Lacla, Lactococcus lactis; Lisin, Listeria innocua; Metja, Methanoccocus jannaschii; Metka, Methanopyrus kandleri; Metma, Methanosarcina mazei; Meslo, Mesorhizobium loti; Mycle, Mycobacterium leprae; Myctu, Mycobacterium tuberculosis; Neucr, Neurospora crassa; Nossp, Nostoc sp.; Prost, Providencia stuartii; Pyrab, Pyrococcus abyssi; Pyrae, Pyrobaculum aerophilum; Ralso, Ralstonia solanaraceum; Sacce, Saccharomyces cerevisiae; Schpo, Schizosaccharomyces pombe; Sinme, Sinorhizobium meliloti; Strco, Streptomyces coelicolor; Strpn, Streptococcus pneumoniae; Sulso, Sulfolobus solfataricus; Sulto, Sulfolobus tokodaii; Synsp, Synechocystis sp.; Theac, Thermoplasma acidophilum; Thema, Thermotoga maritima; Thete, Thermus thermophilus; Vibch, Vibrio cholerae; Xanca, Xanthomonas campestris; Xylfa, Xylella fastidiosa.

Figure 2

Phylogenetic tree of the rhomboid family. The sequences and their regions used to construct the tree are exactly those shown in Figure 1. The color coding and abbreviations are as in Figure 1. The two major eukaryotic subfamilies are denoted as RHO and PARL (see text) and four clusters containing unexpected (from a phylogenetic viewpoint) sets of species are denoted 1-4. The clades that were investigated in the KH test are denoted A through D. Although the tree is shown in a pseudorooted form for convenience, this is an unrooted tree. Internal nodes with at least 70% RELL bootstrap supported are denoted by black circles and nodes with a 50-70% support by blue circles. The posterior probabilities reported by the MRBAYES program are indicated for some key internal branches. Domain architectures are connected to the respective proteins by brackets or lines. The domain key is shown at the bottom of the figure.

Figure 3

Hypothesis-specific constraint tree for the rhomboid family. (a) Hypothesis 1, polyphyletic origin of eukaryotic rhomboids from prokaryotic progenitors. The RHO and PARL subfamilies are denoted; the remaining clusters include prokaryotic rhomboids designated as in Figure 2 (with 'a' added to the GI number). Within each cluster, the branches were collapsed into a multifurcation. (b) Hypothesis 2, monophyletic origin of eukaryotic rhomboids. All eukaryotic and prokaryotic sequences were collapsed into the two respective clusters. The trees are unrooted, although shown in a pseudorooted form.

Figure 4

A hypothetical scenario for the origin and dissemination of the rhomboid family proteases. The figure schematically shows the proposed three stages of evolution of the rhomboid family. In (a), the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage. In (b), the catalytic site of the intramembrane protease evolves, allowing the switch to RIP as the mechanism of the regulatory peptide release. In (c), the emergence of RIP is followed by a burst of HGT. R, regulatory peptide. The transmembrane helices of rhomboid are designated as in Figure 1; their topology in the membrane is based on that proposed in [7]. The catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge-relay system of the protease; possible ancillary catalytic residues are not shown.