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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.
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.
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.
Polytopic transmembrane proteins are, in general, not particularly strongly conserved during evolution. Inspection of the database of Clusters of Orthologous Groups of proteins (COGs)  revealed only one family of such proteins that is represented in most of the sequenced bacterial, archaeal and eukaryotic genomes. The prototype of this family is the rhomboid (RHO) protein from Drosophila melanogaster, a developmental regulator involved in epidermal growth factor (EGF)-dependent signaling pathways [2-4]. Not only were homologs of rhomboid detected in prokaryotes and eukaryotes, but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins, such as transporters [5,6]. Specifically, several polar amino-acid residues are conserved in nearly all members of the rhomboid family, suggesting the possibility of an enzymatic activity. As three of these conserved residues were histidines, it has been hypothesized that rhomboid-family proteins could function as metal-dependent membrane proteases [5,6]. Recently, however, it has been shown that RHO cleaves a transmembrane helix (TMH) in the membrane-bound precursor of the TGFα-like growth factor Spitz, enabling the released Spitz to activate the EGF receptor, and that a conserved serine and a conserved histidine in RHO are essential for this cleavage [7,8]. Thus, it appears that rhomboid-family proteins are a distinct group of intramembrane serine proteases. Altogether, the genome of Drosophila encodes seven RHO paralogs (now designated RHO1-7, with the original rhomboid becoming RHO-1), at least three of which are involved in distinct EGF-dependent pathways, apparently through proteolytic activation of diverse ligands of the EGF receptor [9,10].
The newly discovered intramembrane proteolytic activity of RHO places the rhomboid family within the framework of regulated intramembrane proteolysis (RIP), a new paradigm of signal transduction, which appears to be prominent in all forms of life [11,12]. Under RIP, signaling proteins undergo site-specific proteolysis within TMH, resulting in the release of active fragments, which are the actual effectors in signal tranduction cascades. Until recently, the only characterized cases of RIP in eukaryotes involved presenilin-1, an aspartyl protease, which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid β-precursor protein (AβPP), Notch and Ire1 , and the metalloprotease S2P, which cleaves a TMH in a type-2 transmembrane protein, the sterol-dependent transcription factor SREBP . Notably, S2P has highly conserved bacterial homologs, and the protease domain of presenilins also might be homologous to bacterial and archaeal type IV prepilin peptidases, although, in this case, the sequence similarity is low [14,15].
In the case of the rhomboid family, the existence of homologs of RHO in most prokaryotes is particularly remarkable because animal RHO proteins are involved in signaling pathways that are not found outside metazoa, which seems to make functional conservation in prokaryotes a remote possibility. The only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is AarA from the bacterium Providencia stuartii [16,17]. This protein is involved in the export of a quorum-sensing peptide, a function that, in physiological terms, resembles that of RHO, although the signaling molecules, other than RHO and AarA, are obviously unrelated . In a striking recent development, two independent research groups have shown that several bacterial rhomboid-family proteins, including AarA, can cleave the EGF receptor ligands (Spitz, Keren and Gurken) that are normally cleaved by RHO paralogs [19,20]. The cleavage depended on the conserved serine and histidine residues  and, moreover, transgenic flies that expressed AarA developed a phenotype indistinguishable from that induced by overexpression of RHO, whereas RHO could substitute for AarA in Providencia stuartii . These unexpected findings demonstrated the conservation of a RIP mechanism producing extracellular signals in eukaryotes and prokaryotes. Eukaryotic rhomboid family proteins seem to show considerable functional variability; in particular, cross-talk might exist between different RIP pathways. A distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2, and was accordingly named presenilins-associated rhomboid-like protein (PARL) . The yeast ortholog of PARL has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion .
The near ubiquity of the rhomboid family among bacteria, archaea and eukaryotes, along with the remarkable functional conservation, suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms, with subsequent loss in several lineages. To address this possibility, we performed a detailed phylogenetic analysis of the rhomboid family.
Although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low (around 10-15% identity in the conserved region), the entire superfamily could be retrieved from the protein sequence databases within three iterations of the PSI-BLAST program with a high statistical significance and without any false positives. The conserved core of the rhomboid family consists of six conserved TMHs (Figure (Figure1).1). The predicted catalytic serine is located in TMH5, whereas the predicted catalytic histidine is in TMH7; TMH3 contains two additional histidines and an asparagine, which are conserved in the great majority of the rhomboid-family proteins (Figure (Figure1).1). The roles of these conserved residues are not known, but, given the remarkable evolutionary conservation, it seems likely that they also contribute to catalysis; indeed, it has been shown that the conserved asparagine is required for the cleavage of Spitz by RHO .
When examining the multiple alignment of the rhomboid superfamily proteins, we noticed that several eukaryotic members appear to be inactivated proteases, as indicated by the loss of the predicted catalytic serine or histidine (Figure (Figure1,1, and data not shown); these inactivated forms could be regulators of active rhomboid proteases. Several other proteins lack one or more of the conserved residues in TMH3; it remains unclear whether or not these are active proteases.
Bacterial and archaeal members of the rhomboid superfamily contain six TMH, whereas the eukaryotic members typically have an additional seventh TMH, which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below.
The phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life, but is not necessarily essential for cell function. Rhomboids are missing in the microsporidian Encephalitozoon cuniculi, a eukaryotic intracellular parasite with a highly degraded genome, the archaea Methanothermobacter thermoautotrophicus and Thermoplasma volcanium, and several bacterial species, primarily parasites with small genomes but also species with moderately sized genomes, such as Xylella fastidiosum (see COG0705 at ). In two instances, a representative of the rhomboid family is present in only one of a pair of relatively close genomes (present in T. acidophilum but missing in T. volcanium; present in the spirochete Treponema pallidum but missing in the related bacterium Borrelia burgdorferi), which suggests relatively recent, repeated losses of this gene. Most of the prokaryotic species have a single gene coding for a rhomboid-family protein, although some have two or three paralogs (see COG0705 ); in contrast, eukaryotes show expansion of the rhomboid family, with seven members in Drosophila, and as many as 13 in Arabidopsis.
The multiple alignment of the 6-TMH core of the rhomboid family (Figure (Figure1)1) was employed to construct a phylogenetic tree using the least-squares algorithm with subsequent optimization using the maximum likelihood (ML) method (see Materials and methods). Only the conserved regions including the TMH and short adjacent stretches shown in Figure Figure11 were used as the input for tree building, whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues (except for the Bayesian analysis, which used the complete alignment; see Materials and methods). The alignment used for phylogenetic reconstructions included 87 sequences and 149 aligned sites. The phylogenetic tree of the rhomboid family presents a complex and unexpected picture (Figure (Figure2).2). Neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades. Instead, the eukaryotic rhomboids are split between two major subfamilies, which are positioned in the midst of different prokaryotic branches (Figure (Figure2).2). The first subfamily, which includes six of the seven Drosophila rhomboids, clusters with a distinct prokaryotic assemblage, consisting primarily of Gram-positive bacteria as well as a subset of archaea; this clade is strongly supported by bootstrap analysis (Figure (Figure2).2). The proteins in this group of eukaryotic rhomboids, which we designated the RHO subfamily, typically have an extra TMH added carboxy-terminally to the 6-TMH core; some of these proteins also contain EF-hand calcium-binding domains amino-terminally of the core (Figure (Figure22).
The second eukaryotic subfamily, which we designated the PARL subfamily, after PARL, the human ortholog of Drosophila RHO7 , resides within a large, heterogeneous prokaryotic cluster (Figure (Figure2).2). Within this subfamily, PARL and its orthologs from other animals and from fungi have distinct domain architecture, with an extra TMH added to the amino terminus of the core, whereas the rest have only the core (a carboxy-terminal TMH and a ubiquitin-associated domain are appended in one Arabidopsis protein; Figure Figure2).2). Thus, the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters. Within these two major eukaryotic subfamilies, evolution apparently proceeded by both ancient and more recent duplications. Several lineage-specific expansions of paralogs  are noticeable, in insects, mammals and plants (Figure (Figure22).
Archaeal rhomboids are scattered over the phylogenetic tree, with two major clusters and, in addition, three isolated proteins joining different bacterial branches (Figure (Figure2).2). There is no indication of an affinity between any of the archaeal and eukaryotic rhomboids. Although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages, there are also several clusters that have an odd composition, such as the grouping of proteobacterial and Gram-positive species; some of these clusters are well supported by bootstrap (see clusters 1-4 in Figure Figure22).
Unexpected tree topologies often emerge due to artifacts of phylogenetic analysis methods. This concern is particularly serious for highly divergent families of membrane proteins, such as the rhomboids, in which parallel amino-acid substitutions are likely. Therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests. First, we assessed the robustness of the topology of the tree shown in Figure Figure22 using the Kishino-Hasegawa (KH) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated. Specifically, the KH test was used to evaluate two alternative topologies, in which the RHO and PARL subfamilies formed a clade, and two topologies, in which the RHO subfamily formed a clade with archaeal rhomboids (Figure (Figure22 and Table Table1).1). Each of these alternative topologies had a significantly lower likelihood than the original topology shown in Figure Figure22 (see Table Table11).
In addition, a tree of the rhomboid family was constructed using the Bayesian inference method, which has recently become a practical alternative to the more traditional methods of phylogenetic analysis [24,25]. The tree produced using the MRBAYES package  showed the same major clades as the tree in Figure Figure22 (data not shown); moreover, clustering of the RHO and PARL subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities (Figure (Figure22).
We also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method . The resulting tree contained the same major clades as the trees constructed using ML and MRBAYES; however, the number of parsimony-informative sites was insufficient to obtain high bootstrap support with this approach (data not shown).
We also tested alternative phylogenies using neighbor-joining search with constraint trees . The alternative phylogenies reflected two distinct hypotheses: first, clustering of the RHO and PARL subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in Figure Figure2;2; and second, monophyly of the eukaryotic rhomboids (Figure (Figure3).3). The phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests (Table (Table2).2). The hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly (p < 0.05) worse than the best tree according to each of the tests (Table (Table22).
The concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in Figure Figure22 and, in particular, for the polyphyly of eukaryotic rhomboids.
The phylogenetic tree of the rhomboid family shown in Figure Figure22 and supported by the additional tests described above follows neither the 'standard model' scenario [28,29], with the major split between the archaeo-eukaryotic and bacterial lineages nor the 'mitochondrial' scenario, which postulates acquisition of a gene by eukaryotes from the pro-mitochondrial endosymbiont. Neither can this tree be explained by postulating a small number of lineage-specific gene losses. The parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer (HGT) and lineage-specific gene loss events. In particular, in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms, phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor (LUCA). Instead, the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by HGT, and then lost in some lineages. Both archaea and eukaryotes seem to have acquired rhomboids on several independent occasions. In particular, at least two HGT events seem to have contributed to the origin of eukaryotic rhomboids, one of them yielding the RHO subfamily and the other one the PARL subfamily, with a possible additional HGT in plants (Figures (Figures22,,33).
Given the broad phyletic representation of both subfamilies of eukaryotic rhomboids, both the RHO subfamily and the PARL subfamily must have been acquired through HGT at an early stage of eukaryotic evolution, definitely before the divergence of the major crown-group lineages. This early epoch in eukaryotic evolution is thought to have been dominated by HGT from multiple bacterial symbionts [30,31].
An alternative to this multiple-HGT scenario is that LUCA already had multiple, paralogous rhomboids, which evolved by a series of ancient gene duplications, and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs. Although this cannot be ruled out formally, this hypothesis implies the existence of an elaborate signaling system in LUCA and, accordingly, suggests that LUCA was a complex organism, which might have had as many genes as modern bacteria. Theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of COGs by applying the parsimony principle shows that the complexity of the inferred gene set of LUCA critically depends on the relative rates of gene loss and HGT at the early stages of evolution . A complex LUCA with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of HGT. However, explicit reconstruction of the gene set of LUCA under the assumption of equal rates of gene loss and HGT leads to a hypothetical genome that consists of only around 600 genes but appears to be 'compatible with life', that is, it includes genes responsible for most, if not all, essential cellular functions . We currently believe that this is the most realistic, albeit inevitably imprecise, reconstruction of LUCA's gene set. With respect to the rhomboid family and other families whose phylogenetic trees show similar patterns, this makes the multiple-HGT interpretation the scenario of choice. Further theoretical, comparative-genomic and experimental analyses aimed at determining relative rates of gene loss and HGT will help in a more objective assessment of the validity of this argument.
The multiple-HGT interpretation of the evolutionary history of the rhomboid family, while supported by the above argument, seems, at least at first glance, distinctly counter-intuitive, given that this family is nearly ubiquitous among extant life forms. Indeed, when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [31-33], there is no chance that such a widespread family is not assigned to LUCA. It should be realized, however, that these approaches are inherently probabilistic, and extensive HGT can fool them . For the rhomboid family, the multiple-HGT mode of evolution seems to be particularly plausible. It seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein, probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria. The protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the TMHs (Figure (Figure4).4). This would enable the transition from simple transport to the RIP mode of controlled export of signaling molecules. Emergence of RIP could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed, rather than eliminated, after HGT. In terms of the evolution of sequence itself, the requirements for the conservation of the protease activity apparently 'locked' the rhomboid family in a regime of relatively slow evolution, which ensures significant sequence similarity between all family members (Figure (Figure1).1). The scenario of origin from non-catalytic transporters might potentially apply to other integral membrane enzymes, including intramembrane proteases involved in RIP, such as presenilins and their homologs [14,15] and the archaeo-eukaryotic signal peptide peptidase .
The rhomboid family might be the most widespread and conserved group of integral membrane proteins. In and by itself, this would suggest that this family is part of the gene repertoire of LUCA. However, phylogenetic analysis suggests a different scenario, one of emergence in a bacterial lineage with subsequent multiple, independent HGT events and gene losses. Although caution is due in the evolutionary interpretation of phylogenetic trees for large families, particularly when membrane proteins with a relatively small number of conserved positions, such as the rhomboids, are involved, the multiple-HGT scenario seemed to be supported by several methods of tree analysis and statistical tests.
Eukaryotes probably acquired their two major rhomboid subfamilies, RHO and PARL, as the result of two independent, early HGT events. These events, which might have introduced RIP as a means of intercellular communication, could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery . Subsequent evolution of rhomboids in eukaryotes proceeded by lineage-specific expansion of paralogs  followed by diversification through the addition of an extra TMH in different positions relative to the catalytic core, some limited domain accretion (see Figure Figure2)2) and sequence divergence.
Phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms, particularly LUCA. Although most of the (nearly) ubiquitous protein families probably do derive from LUCA, explicit phylogenetic analysis is required to ascertain this in each case.
The nonredundant (NR) protein sequence database at the National Center for Biotechnology Information (NIH, Bethesda) was searched iteratively using the PSI-BLAST program with multiple starting queries . PSI-BLAST was normally run with expectation (E) value of 0.01 as the cut-off for inclusion of sequences into the position-specific scoring matrix. Multiple alignments of protein sequences were constructed using the ClustalW program  and manually adjusted on the basis of the examination of PSI-BLAST search outputs and the superposition of the predicted TMHs, which were identified using the programs TMpred  and TMAP .
Phylogenetic trees were built using the least-squares method  implemented in the FITCH program of the PHYLIP package , with subsequent local rearrangement using the PROTML program of the MOLPHY package to obtain the maximum likelihood tree . The reliability of the tree topology was assessed using the RELL (resampling of estimated log-likelihoods) bootstrap method of MOLPHY, with 10,000 replications . Alternative placements of selected clades in maximum-likelihood trees were compared by using the rearrangement optimization method (Kishino-Hasegawa test) as implemented in the ProtML program [43-45]. Maximum parsimony trees were constructed using the heuristic search option of PAUP* . In addition, trees were constructed by Bayesian inference using the Markov chain Monte Carlo method as implemented in the MRBAYES package [24,26]. The complete alignment information, including columns with gaps, was used for the MRBAYES analysis.
Constraint trees for phylogenetic hypothesis testing were generated using the TreeView program . Constraint trees were imported into PAUP*  and subjected to neighbor-joining search to generate the phylogenies corresponding to alternative hypotheses. These phylogenies were compared using the KH , Templeton (Wilcoxon signed-ranks)  and Winning-sites (sign)  tests implemented in PAUP*.
L.P. is supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
A previous version of this manuscript was made available before peer review at http://genomebiology.com/2002/3/11/preprint/0010/