SAMPylation was recently introduced by the Maupin-Furlow group at the University of Florida as a novel protein conjugation system in archaea with parallels to the ubiquitin conjugation system. SAMPylation has gotten quite a bit of attention. Attachment of SAMP1 and SAMP2 (members of the ThiS/MoaD clade, which is the basal lineage of the larger ubiquitin-like clade of beta-grasp domains) to a target protein via an E1-like ligase might be interpreted as representing a rudimentary form of the classical eukaryotic Ubiquitin (Ub) and Ubiquitin-like (Ubl) conjugation system, which attaches Ub/Ubl domains to targets via a tri-ligase enzyme cascade initiated by an E1-like ligase and completed by the E2 and E3 ligases. In forthcoming articles, we review origins of SAMPylation (among other issues) in the context of other prokaryotic protein tagging systems and the eukaryotic ubiquitin system. Given the burgeoning interest in SAMPylation, however, we’ll preview these articles with a set of extended thoughts specific to the origins of SAMPylation.
1) Phyletic distributions and phylogenetic affinities of SAMP1/SAMP2
The SAMP2 protein is clearly a member of a small branch of the ThiS clade, which is restricted to euryarchaeota, with substantial representation in the haloarchaea as well as some methanoarchaea. The SAMP1 protein, on the other hand, is clearly a member of the MoaD clade; however, the phylogenetic relationships of the MoaD clade are complicated by the repeated occurrence of gene duplications and horizontal gene transfer (HGT) events. Recently, phylogenetic relationships between bacterial genomes have been described in terms of a “thicket” rather than a linear tree due to rampant genome duplication and exchange through HGT. Analogously, one can think of the MoaD gene family as the MoaD gene “thicket”. This leads to a poor picture of the divisions among the multiple functional roles ascribed to the MoaD family at large, including the incursion of a protein tagging role in SAMP1.
2) Multiple functional roles for the MoaD homolog thicket and the ThiS clade
Despite difficulties in assigning function to uncharacterized members of the MoaD family, a wide range of functional roles have been identified for MoaD-like proteins. In fact, it is no longer appropriate to view the MoaD and ThiS families exclusively as sulfur carriers for molybdenum and thiamine cofactor biosynthesis, respectively. As well as roles for both families in SAMPylation, they are also involved in sulfur transfer during siderophore-like compound and cysteine biosynthesis and tRNA thiolation. Our group predicts additional functions for these families in tungsten cofactor biosynthesis and perhaps a tagging role in recruitment to the ClpAP complex via association with a ClpS domain. As an added twist, some MoaD/ThiS domains have the demonstrated ability to carry out more than a single functional role. The second study on SAMPylation from the Maupin-Furlow group demonstrates that in Haloferrax, the SAMP1 protein is indispensible for MoCo/WCo biosynthesis to the point that the primary role for SAMP1 is indeed be akin to the classical MoaDs to which it is closely related. Meanwhile, SAMP2 appears to be important in tRNA thiolation as well as conjugation (Comparable to Urm1). Notably, all of these functional roles are thought to be performed in conjunction with an E1-like ligase domain.
The takeaway from these observations is that the ThiS/MoaD-E1 enzyme combination has proven to be an extremely adaptable platform for acquisition of novel functional roles, primarily in the context of sulfur incorporation but also for protein tagging (as seen now in SAMPylation, Urmylation, and Ubiquitination).
3) Independent emergence of multiple protein tagging systems in prokaryotes
SAMPylation is the latest in a string of newly-discovered prokaryotic tagging systems, perhaps most well-known is the co-translational tmRNA-based peptide tag. More recently the PUPylation system has been discovered. PUPylation involves attachment of the Pup protein to target proteins via the PafA ligase, unrelated to the SAMP/Ub tag/ligase counterparts. Most relevant to this discussion is the phyletic distribution of PUPylation, detailed in our past work. PUPylation-like systems are sporadically present in a range of bacterial lineages; however, these systems very clearly emerged first in actinobacteria before spreading to other lineages via HGT. This limited distribution very much resembles the distribution of SAMP2ylation.
In addition to PUPylation, several other tagging systems are known to prokaryotes: 1) our group has pioneered in the identification of ubiquitin-like systems containing the Ub tri-ligase (E1, E2, and E3-like ligase) complement in bacteria, while another group recently identified a similar system after sequencing the archaeon Candidatus Caldiarchaeum subterraneum. 2) N-end rule arginyl/leucyl ligation. 3) Convergently-emergent protein tagging systems in diverse prokaryotes predicted in a separate paper from our group. 4) Predicted conjugation of certain members of the YukD family. Given this list, it appears tagging systems have emerged multiple times in prokaryotes. The mode of emergence of many of these tagging systems also shares commonalities, having frequently emerged first in a particular lineage of prokaryotes, only to be transferred later to additional sporadic representatives of diverse lineages via HGT.
4) SAMPylation is condition-specific
To this point, SAMP2 has been demonstrated to form conjugates with substrate proteins (and itself) only in response to two conditions: nitrogen and oxygen depletion. SAMP1 conjugation is largely restricted to substantial nitrogen depletion.
Summary and outstanding questions
The above observations begin to clarify the relationship between SAMPylation and other prokaryotic tagging systems. Phyletic distributions and peripheral or condition-specific functional roles seem to suggest prokaryotic tagging systems in general have secondarily emerged within differentiated lineages, with the ThiS/MoaD/Ub-E1 functional connection seemingly particularly suited for acquisition of tagging roles. This description is particularly apt for the SAMP2ylation system which appears to be a genuine, condition-specific adaptation restricted to a small group of archaea. One key consequence of this observation: it is highly unlikely that SAMP2ylation represents a precursor for classical Ubiquitination. The greater mystery, however, lies with SAMP1ylation. At one extreme, SAMP1ylation could occur in all MoaD family members across bacteria and archaea, directing tagged proteins for proteosomal degradation. Given the broad distribution of MoaD and the relatively restricted distribution of core proteasomal subunits this seems questionable, particularly for bacterial MoaD-like domains. At the other extreme, MoaD family members are only conjugated in genomes capable of performing SAMP2ylation; a scenario implying conjugation of SAMP1 under certain conditions was adapted from the SAMP2ylation apparatus. Addressing key points outlined below surrounding SAMP1ylation would help immensely in clarifying the Ubiquitination/SAMPylation relationship.
Q1) What is the extent to which SAMP1 is used as a modifier?
The MoaD clade thicket (of which SAMP1 is a member) has been adapted to a vast and varied range of functional roles. Further analysis of SAMP1ylation in the MoaD clade is needed to define the precise conditions and functional contexts in which SAMP1ylation, if any, is observed amongst the set of all MoaD clade proteins.
Q2) Is there a relationship between the extreme conditions required to initiate SAMPylation and the likely functional roles for SAMP1 (and SAMP2)?
SAMP1, and not SAMP2, is linked to proteosomal degradation due to the accumulation of SAMP1ylated substrates in proteosomal subunit mutant strains. Why does extreme nitrogen depletion signal for sudden degradation of massive amounts of cellular protein? As an alternative theory, could extreme nitrogen depletion cause the UbaA protein to become more “promiscuous” in recognition of ThiS/MoaD-like substrates for conjugation? In a similar vein, the function of SAMP2ylation is still not entirely clear, although it has been linked to conjugation of proteins predicted to be involved in tRNA thiolation. Is SAMP2ylation acting as a negative feedback loop regulating the primary sulfur-carrier functional role for SAMP2 in tRNA thiolation? It remains to be seen if these conjugates are subject to deSAMPylation by some of the JAB domain proteins encoded in these genomes. This might better help in clarifying the above questions.
Q3) What happens when there is overlap between SAMP1ylation and other potential tagging systems, i.e. in bacteria?
If all MoaD-like proteins are capable, to some degree, of being conjugated to other proteins under the right conditions, this would result in the overlap of SAMPylation with other protein tagging systems including some specifically involved in targeting proteins for proteasomal-based destruction. This is most notable in the actinobacteria, where unlike the overlap between PUPylation and prokaryotic ubiquitin-like conjugation systems, which appear to occupy distinct functional niches (discussed in another post here), SAMP1ylation would directly functionally overlap with PUPylation.
The recent characterization of diverse tagging systems strongly suggests protein ligation emerged several times in prokaryotic evolution. Several of these systems are known or predicted to be coupled to proteasomal degradation, specifically suggesting multiple, distinct inventions of protein tagging-directed degradation. Many tagging systems appear to have emerged later in prokaryotic evolution within certain terminal branches of the bacterial tree followed by wider dissemination to additional lineages, in varying degrees, via HGT. In particular, more research into the generality of of SAMP1ylation should be investigated in greater detail across bacteria and archaea. In the absence of support of for its generality, and given the lineage-specific nature of SAMP2, the current balance of evidence, cannot rule out, and might in fact support a scenario where SAMPylation via E1-only conjugation systems represents a separate development. This type of conjugation, especially SAMP2lyation, might have emerged in parallel to the elaboration of more complete prokaryotic ubiquitination systems that contain just E2 or both E2 and E3 ligases, rather than being precursors of these systems.
On the other hand, evidence from eukaryotes for conjugation of Urm1 suggests that indeed a protein conjugation capability might have been latent right from the ancestral Ub-like beta-grasps domains that participated in ancient biosynthetic sulfur insertion functions. It is possible that this capability was an “unintended” consequence of the chemistry of sulfotransfer, primarily coming to fore under certain special conditions, not necessarily in a functional sense – i.e., it might be viewed as a Gouldian spandrel. However, in certain organisms it was exapted to different degrees and functionally “channelized” as a protein tag. Indeed, protein tags are a necessary prerequisite for targeted protein degradation and provide an additional advantage a regulatory mechanism – these provide a “niches” into which the Ubl-E1 dependent conjugation systems could diversify as protein tags. We had earlier discussed how distinct protein tagging systems for targeting proteins for degradation are basal and nearly universal across two of the three superkingdoms of life – the tmRNA-based system in bacteria and the Ub-based system in eukaryotes. Only the archaea, which ancestrally share the proteasome with eukaryotes seemingly lack such a system. From this viewpoint, evidence for cognates of SAMPylation across archaea, based on the more widely distributed MoaD clade, certainly need to be further investigated.
Additional Reading: Click on the pubmed ids to access the references
- Experimental characterization of SAMPlylation in Marlin-Furlow lab, 20054389, 21368171
- Discovery of PUPylation in the Darwin lab 18832610
- Evolutionary history of the PUPylation system 18980670
- Evolution of Ubl pathways in prokaryotes 16859499 21547297 21169198
- Higher-order relationships between five-stranded beta-grasp domain-containing protein families including ThiS, MoaD, Urm1, Ub/Ubl 17605815
- Functional linkages between E1 and ThiS/MoaD, Ub/Ubl 19089947