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10 July 2019

Rhodanese-Like Domain Protein UbaC and Its Role in Ubiquitin-Like Protein Modification and Sulfur Mobilization in Archaea


Ubiquitin-like protein (Ubl) modification targets proteins for transient inactivation and/or proteasome-mediated degradation in archaea. Here the rhodanese-like domain (RHD) protein UbaC (HVO_1947) was found to copurify with the E1-like enzyme (UbaA) of the Ubl modification machinery in the archaeon Haloferax volcanii. UbaC was shown to be important for Ubl ligation, particularly for the attachment of the Ubl SAMP2/3s to protein targets after exposure to oxidants (NaOCl, dimethyl sulfoxide [DMSO], and methionine sulfoxide [MetO]) and the proteasome inhibitor bortezomib. While UbaC was needed for ligation of the Ubl SAMP1 to MoaE (the large subunit of molybdopterin synthase), it was not important in the formation of oxidant-induced SAMP1 protein conjugates. Indicative of defects in sulfur relay, mutation of ubaC impaired molybdenum cofactor (Moco)-dependent DMSO reductase activity and cell survival at elevated temperature, suggesting a correlation with defects in the 2-thiolated state of wobble uridine tRNA. Overall, the archaeal stand-alone RHD UbaC has an important function in Ubl ligation and is associated with sulfur relay processes.
IMPORTANCE Canonical E2 Ub/Ubl-conjugating enzymes are not conserved in the dual-function Ubl systems associated with protein modification and sulfur relay. Instead, the C-terminal RHDs of E1-RHD fusion proteins are the apparent E2 modules of these systems in eukaryotes. E1s that lack an RHD are common in archaea. Here we identified an RHD (UbaC) that serves as an apparent E2 analog with the E1-like UbaA in the dual-function Ubl sampylation system of archaea. Unlike the eukaryotic E1-RHD fusion, the archaeal RHD is a stand-alone protein. This new insight suggests that E1 function in Ubl pathways could be influenced by shifts in RHD abundance and/or competition with other protein partners in the cell.


In archaea, ubiquitin-like (Ubl) modification targets proteins for transient inactivation and/or degradation in pathways associated with regulation, metabolism, and other processes (13). Similarly to ubiquitin (Ub), archaeal Ubls are β-grasp fold proteins with C-terminal di-Gly motifs that are covalently attached to the lysine residues of protein targets (46). To initiate the covalent modification, an E1-like enzyme adenylates the C terminus of the Ub/Ubl and forms an E1∼Ub/Ubl thioester intermediate (7, 8). In eukaryotic ubiquitination, an E2 enzyme accepts the Ub from the E1∼Ub thioester, and an E3 ligase guides the Ub to its protein target. In contrast, the steps downstream of the E1∼Ubl thioester formation are not as well defined in archaea. Evolutionary precursors of enzymes involved in ubiquitination are detected in archaea, including short E2- and E3-like gene homologs in Aigarchaeota and Asgard archaea (9, 10). In addition, methionine sulfoxide reductase A (MSRA), which is related by convergent evolution to the MSRB-fold substrate receptor of the E3 ligase Cereblon, is required for Ubl modifications in Euryarchaeota (11).
Ub/Ubl ligation systems have prototypes in sulfur relay pathways that use β-grasp fold Ubls as sulfur carriers to form sulfur-containing biomolecules, such as molybdenum cofactor (Moco), thiamine, and 2-thiolated tRNA (12, 13). In these pathways, an E1-like enzyme activates the C terminus of the Ubl through an adenylation reaction that resembles the first step of Ub/Ubl ligation (see, e.g., references 14 to 16). However, in contrast to Ub/Ubl ligation, the C terminus of the Ubl is converted from an adenylate to a thiocarboxylate that serves as a sulfur donor for the sulfur-containing biomolecules to be synthesized (16).
The source of sulfur for the Ubl thiocarboxylation is often a cysteine persulfide formed on a rhodanese-like domain (RHD) (17). In Moco biosynthesis, the cysteine persulfide that supplies the sulfur to the Ubl is derived from the active site of a C-terminal RHD of the E1-like MOCS3/Cnx5 in humans/plants or a stand-alone RHD (YnjE) in bacteria such as Escherichia coli (18, 19). While Saccharomyces cerevisiae Uba4 is a homolog of MOCS3/Cnx5, this model yeast does not generate Moco (20). However, the RHD of all three eukaryotic E1-like homologs (MOCS3/Cnx5/Uba4) does supply sulfur for the thiocarboxylation of the Ubl Urm1/Urm11 in the 2-thiolation of wobble uridine tRNA (15, 21); the tandem RHD TUM1 is apparently needed upstream of this reaction (22). The eukaryotic E1-fused RHD is also needed for urmylation (i.e., the Ubl ligation of Urm1 to protein targets) (23) and thus is hypothesized to be a primordial E2 module in Ubl modification (24, 25). Consistent with an association of RHDs in Ubl pathways, RHDs are also found to promote protein-protein interactions with E3 ligases, as demonstrated by the atomic-resolution docking of the RHD of the human Ub-specific protease 8 (Usp8) to the E3 ligase NFDP1 (26).
In archaea, E1-like enzymes, such as Haloferax volcanii UbaA, are central hubs that activate the Ubls for ligation to protein targets and for sulfur mobilization (8). UbaA is required for the Ubl small archaeal modifier proteins (SAMPs) to serve as protein modifiers (4, 5) and to carry sulfur to biomolecules (8). SAMP1 carries sulfur to form Moco, based on its binding to MoaE (the large subunit of molybdopterin [MPT] synthase) and its requirement for Moco-dependent dimethyl sulfoxide (DMSO) reductase activity (3, 8, 27). In contrast to SAMP1, SAMP2 binds NcsA (a Tuc1/Ncs6 tRNA 2-thiolation protein homolog) and is required (along with NcsA) for the thiolation tRNA with wobble uridine in the anticodon position [e.g., tRNALys(UUU)] (8, 28). Whether an archaeal RHD is associated with Ubl pathways remains to be determined. Like most archaeal E1s that cluster to the Ub-activating enzyme (IPR035985) superfamily, H. volcanii UbaA is not fused to an RHD. Furthermore, RHD UbaB encoded in genome synteny with UbaA is not needed for Ubl ligation or sulfur mobilization to form Moco or thiolated tRNALys(UUU) (8).
In the present study, the stand-alone RHD HVO_1947 (named UbaC) of H. volcanii was found to bind the E1 UbaA and to be required for Ubl modification and for functions associated with Ubl sulfur relay. UbaC and its conserved active-site cysteine (C64) were (i) required for DMSO respiration/reductase activity correlated with Moco biosynthesis, (ii) important for survival at elevated temperature, which is indicative of a functional wobble uridine tRNA thiolation pathway, and (iii) critical for the formation of Ubl ligation products, with marked exception of the SAMP1 conjugates induced by oxidative stress. Our results provide insight that stand-alone RHDs, such as UbaC, can serve in sulfur relay pathways and are suggested to be E2-like modules in Ubl ligation. Thus, regulation of the protein-protein interactions and/or levels of stand-alone RHDs and E1s may influence cellular Ubl systems.


Genome comparison of archaeal RHDs.

RHD protein fusions and stand-alone proteins are widespread in archaea but poorly understood. Of the seven RHD proteins of H. volcanii, two are tandem RHDs (HVO_0025 and HVO_0024), three are RHDs fused to a C-terminal metallo-β-lactamase domain, and two are stand-alone RHDs (UbaB and UbaC) (Fig. 1). Of these RHDs, UbaB is in genome synteny with the E1-like UbaA, suggesting that it may be associated with the Ubl systems; however, UbaB is not required for Ubl modification or sulfur mobilization to form Moco or thiolated wobble uridine tRNA, based on a previous study (8).
FIG 1 H. volcanii rhodanese-like domain (RHD) proteins. Gene locus tag and UniProt accession numbers are indicated on the left. Colored boxes represent the protein domains determined by InterPro protein sequence analysis and classification ( Conserved active-site cysteine residues of the RHDs (in red) and conserved cysteine residues and an HXHADH motif of the metallo-β-lactamase domains are indicated.
To further examine the relationship of RHDs to Ubl systems, two sequence similarity networks (SSNs) were generated. First, archaeal E1s (including those with C-terminal RHDs) were analyzed for their similarity in amino acid sequence and protein domain architecture (Fig. 2). The most common domain fused to the archaeal E1s was the RHD, followed by domains related to JAB/MPN metalloproteases that cleave Ub/Ubl isopeptide linkages (3, 27, 29) and β-grasp fold proteins (Ub and ThiS) (Fig. 2). Most archaeal E1s, however, were stand-alone proteins, including H. volcanii UbaA, which shared close relationship to the E1s fused to RHDs. To further understand the E1-RHD relationship, a second SNN was generated by comparing the RHDs extracted from the eukaryotic E1s (MOCS3/Cnx5/Uba4) and archaeal arCOG02021 (Fig. 3A). When examining the H. volcanii RHDs in this SSN configuration, UbaB/C and the metallo-β-lactamase-fused RHDs were found in distinct clusters connected by edges to the central cluster of stand-alone and E1-fused RHDs (Fig. 3A). The H. volcanii tandem RHDs (HVO_0024 and HVO_0025) were distinct and did not appear to be related to the E1-RHDs (Fig. 3A). Alignment of UbaB/C to the C-terminal domain of the eukaryotic E1-RHD fusions MOCS3/Uba4/Cnx5 revealed the key active-site cysteine that carries the persulfidic sulfur is conserved (Fig. 3B). UbaB was also found to share 36% identity with UbaC (Fig. 3C, left), with both proteins having predicted three-dimensional (3D) structures related to the human MOCS3 RHD X-ray crystal structure (Fig. 3C, middle and right). Thus, UbaB/C are related to each other and to E1 RHDs and have a conserved cysteine predicted to be modified by a persulfide group (3032).
FIG 2 Sequence similarity network (SSN) of archaeal E1 homologs of the IPR035985 superfamily. Stand-alone E1s are indicated by circles. Extra protein domains fused to the E1s are indicated by colored triangles. The close relationship of H. volcanii UbaA to E1-RHD fusion proteins is noted. Nodes represent proteins, with those of similar sequence clustered by edges (gray lines).
FIG 3 Comparison of RHDs from stand-alone proteins and protein fusions. (A) SSN of RHD domains extracted from arCOG02021, MOCS3 (Hs, human; UniProtKB accession no. O95396), Cnx5 (At, Arabidopsis thaliana; accession no. Q9ZNW0), and Uba4 (Sc, S. cerevisiae; accession no. P38820). Nodes represent proteins, with those of similar sequence clustered by edges (gray lines). (B) Schematic alignment of UbaA (accession no. D4GSF3), UbaB (accession no. D4GSF4), and UbaC (accession no. D4GTH6) of H. volcanii (Hv) with MOCS3, Uba4, Cnx5, and MoeB (Ec, E. coli; accession no. P12282). Consensus site residues are shaded in gray. (C) Comparison of RHDs by multiple amino acid sequence alignment (left), 3D modeling (middle), and zoom-in of active-site loop residues in the 3D structure (right). _N, N-terminal domain. _C, C-terminal domain. The RHD sulfurtransferases EcGlpE (accession no. P0A6V5) and EcSseA (accession no. P31142) are also included for comparison.

UbaC is required for anaerobic DMSO respiration.

To determine the archaeal RHD required for Moco biosynthesis, a series of H. volcanii RHD mutants (ΔubaB, ΔubaC, Δhvo_0024, and Δhvo_0025) was generated and compared to parent strain H26 cells in terms of DMSO respiration. H. volcanii does not grow anaerobically if DMSO is excluded from the medium, and it requires MPT synthase subunit homologs (MoaE and SAMP1) and the E1-like UbaA for DMSO reductase activity (4). After 3 days of incubation at 42°C, the ΔubaB and the tandem RHD (Δhvo_0024 and Δhvo_0025) mutants displayed growth comparable to that of the parent (H26) when supplied with DMSO as the terminal electron acceptor (Fig. 4A). In contrast, little to no growth was observed for the ΔubaC mutant under these conditions (Fig. 4A). The Δsamp1 and ΔubaA mutants were also unable to grow on DMSO (Fig. 4A), as observed in a previous study (8). When ubaC was expressed in trans, the ΔubaC mutant was restored for anaerobic respiration on DMSO, revealing that the effect was specific to ubaC. The conserved active-site cysteine (C64) of the UbaC RHD loop was required, as the UbaC C64S variant did not complement the ΔubaC mutation (Fig. 4A). While UbaC shares amino acid sequence identity with UbaB, presumed overexpression of UbaB from a constitutive rRNA P2 promoter did not restore the ability of the ΔubaC mutant to respire on DMSO (Fig. 4A). Inclusion of tungstate, a known antagonist of molybdate insertion into the pterin ring during Moco maturation in E. coli (33), prevented all H. volcanii strains from respiring DMSO (Fig. 4A). This effect could be reversed for the H. volcanii parent (H26) and ΔubaC mutant trans-expressing ubaC strains by including molybdate in the medium (Fig. 4A). Thus, tungstate had an antagonistic effect on DMSO respiration that could be overcome by molybdate supplementation. The ΔubaC mutant and its ubaC C64S trans-expression strain did not respire on DMSO under all conditions examined (Fig. 4A). Furthermore, all strains grew in the presence of oxygen even when supplemented with tungstate (Fig. 4A), revealing that the shift in terminal electron acceptor from oxygen to DMSO was important for the phenotypes observed. Collectively, these results reveal that UbaC and its active-site cysteine (C64) are required for DMSO respiration and do not share redundant functions with UbaB in this pathway.
FIG 4 UbaC is important for DMSO respiration (A), DMSO reductase activity (B), and growth at elevated temperature (C). (A) Cell growth under DMSO respiration and aerobic conditions with medium supplemented with tungstate (W) and/or molybdate (Mo) as indicated. (B) DMSO reductase activity of H. volcanii strains as indicated. (C) Serial dilution of cells spotted on ATCC 974 plates and grown at 42°C and 50°C. EV, empty vector (pJAM202c). *, C-terminal Strep-tag II tag.

UbaC is needed for DMSO reductase activity.

To further understand the role of UbaC in DMSO respiration, the H. volcanii strains were analyzed for DMSO reductase activity, which requires Moco (34, 35). To test for this activity, clarified lysates were assayed for the oxidization of reduced methyl viologen in the presence of DMSO. By this approach, DMSO reductase activity was detected in the parent (H26) but not in the ΔubaC mutant (Fig. 4B). Furthermore, ectopic expression of UbaC restored the DMSO reductase activity of the ΔubaC mutant, while the empty-vector control and the UbaC C64S mutant did not (Fig. 4B). Thus, ubaC was important for DMSO reductase activity, consistent with its putative role in Moco biosynthesis.

ΔubaC mutants display reduced growth at elevated temperature.

Hypothiolation of wobble uridine tRNA is correlated with reduced translational fidelity and impaired growth at elevated temperature (8, 28, 36, 37). To indirectly address the role of the archaeal RHDs in tRNA thiolation, the H. volcanii RHD mutants were examined for growth at 50°C compared to the 42 to 45°C optimum. The ΔubaB, Δhvo_0024, and Δhvo_0025 RHD mutants were found to grow similarly to the parent H26 at all temperatures examined (Fig. 4C). In contrast, the ΔubaC mutant was impaired for growth at 50°C (but not at 42°C) (Fig. 4C), having a growth phenotype that corresponded to those of ΔubaA and ΔncsA mutants deficient in the 2-thiolation of wobble uridine tRNA (8, 28). Growth of the ΔubaC mutant at 50°C was restored by ectopic expression of ubaC but not ubaC C64S (Fig. 4C). Thus, UbaC and its conserved cysteine (C64) were found to be important for growth at elevated temperature, which may be correlated with tRNA thiolation.

UbaA-UbaC interaction examined by pulldown assay.

The observed impact of UbaC on cellular functions associated with sulfur mobilization suggested that this RHD may physically associate with UbaA in H. volcanii. To examine this potential interaction, a pulldown assay was performed using UbaC–Strep-tag II as the bait. Untagged UbaC was included as a negative control to rule out any nonspecific proteins that may bind the affinity column. UbaA (encoded from the H. volcanii genome) was found to copurify with UbaC–Strep-tag II in a specific manner (Fig. 5). This pulldown experiment provided evidence that UbaA and UbaC interact in the cell.
FIG 5 UbaC and its association with the E1-like UbaA in H. volcanii. Cell lysate of the parent strain (H26) that expressed UbaC or UbaC–Strep-tag II (*) was used as input (lanes 1 and 2). Proteins associated with UbaC–Strep-tag II were enriched by pulldown using Streptactin resin (lanes 4). UbaC served as a control (lane 3) to assess nonspecific proteins (**) detected by immunoblotting (IB) analysis. CBB, Coomassie blue staining of total protein.

UbaC is required for specific Ubl modifications.

The physical association of UbaC with UbaA suggested that UbaC may be required for Ubl modification of protein targets, similarly to the RHD of the Uba4 E1-RHD fusion in urmylation. Thus, the ΔubaC mutation was analyzed for its impact on (i) the covalent attachment of the Ubl SAMP1 to MoaE (3, 5, 8) and (ii) the accumulation of Ubl modifications after exposure of cells to the oxidizing agents DMSO, methionine sulfoxide (MetO), and NaOCl and the proteasome inhibitor bortezomib (5, 11, 38). Ubl modification by SAMP1, SAMP2, and SAMP3 was monitored by individually Flag tagging the SAMPs. The parent H26 and the ΔubaA (E1-like) mutant were included for comparison, with the latter impaired in all Ubl modifications examined (7, 8). By this approach, ubaC was found to be important for the covalent modification of MPT synthase (SAMP1 linked to MoaE). The covalently modified MPT synthase that migrated at 50 kDa was not apparent in the ΔubaC mutant (Fig. 6A and B, lanes 1 versus 4), which instead accumulated Ubl (SAMP1) linkages at 37 and 60 kDa (Fig. 6A, lane 4) that migrated similarly to the Ubl linkages detected when UbaA–Strep-tag II was ectopically expressed (Fig. 6A, lane 5). These alternative Ubl linkages were speculated to be UbaA modified by SAMP1, based on this close relationship in SDS-PAGE migration (Fig. 6A, lane 4 versus 5) and our previous finding by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis that UbaA is autosampylated at K87 and K157 (7). UbaC was not needed for formation of the SAMP1 Ubl linkages that were induced by DMSO (Fig. 6A, lane 10) or MetO (Fig. 6B, lane 9). Contrastingly, most of the SAMP2 Ubl linkages that accumulated in the presence of an oxidizing agent and/or proteasome inhibitor were impaired in the ΔubaC mutant (Fig. 7A, lanes 3, 11, 19, and 25). Note that of the three H. volcanii SAMPs, only SAMP2 appears to function like eukaryotic Ub in targeting proteins for proteasome-mediated proteolysis (1, 5). In addition to SAMP2, the oxidant-induced SAMP3 Ubl linkages were also abolished in the ΔubaC mutant (Fig. 7B, lanes 3 and 10). Ectopic expression of ubaC was found to restore the Ubl SAMP2/3 modifications in the ΔubaC mutant (Fig. 7A, lanes 4, 12, 20, and 26, and B, lanes 4 and 11); UbaC64S was not functional in this restoration (Fig. 7B, lane 8). Consistent with a role in stress-induced Ubl modifications, UbaC and its active-site C64 were important for cell survival after exposure to NaOCl similarly to UbaA (Fig. 8). Thus, UbaC and its conserved active-site cysteine (C64) not only are important for the Ubl SAMP2/3 linkages that accumulate after exposure to oxidant and proteasome inhibitor but also are needed for cell survival during stress, such as exposure to the potent oxidant hypochlorite generated during NaOCl treatment.
FIG 6 UbaC is important for covalent modification of molybdopterin synthase (SAMP1-MoaE). Ubl SAMP1 conjugate abundance was analyzed by immunoblotting of lysate after growth of cells under uninduced and induced conditions using 100 mM dimethyl sulfoxide (+ DMSO) (A) or 25 mM methionine sulfoxide (+ MetO) (B) as indicated. **, N-terminal Flag tag; *, C-terminal Strep-tag II tag; EV, empty vector (pJAM202c).
FIG 7 UbaC is important for SAMP2-type (A) and SAMP3-type (B) Ubl modifications. H. volcanii strains were exposed to the mock control (uninduced), 100 mM dimethyl sulfoxide (+ DMSO), 25 mM methionine sulfoxide (+ MetO), 100 μM proteasome inhibitor (+ bortezomib), and 20 mM NaOCl (+ NaOCl) as indicated. Cells were harvested and analyzed for Ubl modifications by immunoblotting. **, N-terminal Flag tag; *, C-terminal Strep-tag II tag; EV, empty vector (pJAM202c).
FIG 8 UbaC is important for survival after oxidative stress. Cells were exposed to NaOCl and then incubated on ATCC 974 plates as indicated. EV, empty vector (pJAM202c). *, C-terminal Strep-tag II tag.


Here the RHD protein UbaC (HVO_1947) was found to be intimately associated with the E1 UbaA and to be required for Ubl modification and sulfur relay pathways in H. volcanii (see the model proposed in Fig. 9). UbaC copurified with UbaA, suggesting that a multisubunit complex(es) catalyzes these processes. UbaC was essential for DMSO respiration and DMSO reductase activity, suggesting its importance in Moco biosynthesis. UbaC was also found to be important for growth at elevated temperature, much like UbaA, NcsA, and SAMP1, which are needed for the thiolation of wobble uridine tRNA (8, 28). These properties of UbaC, in addition to the finding that the conserved active-site C64 was needed for these functions, were all consistent with a role of this RHD in carrying sulfur as a persulfide at C64 for donation to form Moco and thiolated tRNA. This same cysteine residue (UbaC C64) was required for detecting the Ubl linkages that formed in the cell, similarly to the RHD of Uba4 needed for urmylation (23).
FIG 9 Models of Ubl Urm1 thiocarboxylation in eukaryotes (e.g., S. cerevisiae) (A) compared to Ubl thiocarboxylation and protein ligation in archaea (e.g., H. volcanii) (B). The sulfur that is relayed is highlighted in red. The E1-like and RHD domains/proteins are colored in blue and orange, respectively. Panel A is according to reference 21. Uba4 is a fusion of E1-like and RHD domains. Uba4 catalyzes the adenylation of the C-terminal glycine of the Ubl Urm1 (step 1). The Nfs1-Isd11 cysteine desulfurase complex cooperates with the tandem RHD Tum1 (YOR251C) to relay sulfur from free cysteine to the active-site cysteine of the Uba4 RHD (step 2). The Uba4 E1-like active-site cysteine attacks the Ubl (Urm1)∼adenylate to form a thioester intermediate (step 3). The Uba4 RHD persulfide sulfur attacks this thioester and forms an acyl persulfide with the Ubl (step 4). Reductive cleavage regenerates Uba4 and releases thiocarboxylated Ubl (Urm1) (step 5) for use in sulfur mobilization to form thiolated tRNA (step 6). In panel B, archaea such as H. volcanii are proposed to use the stand-alone E1-like UbaA and RHD UbaC to mediate Ubl protein ligation and mobilize sulfur to form Moco and thiolated tRNA. UbaA alone (independent of other factors such as UbaC and MSRA) binds ATP and the Ubls (SAMPs) (step 1a), catalyzes Ubl adenylation (step 1b), forms a UbaA∼Ubl thioester (step 3a), and undergoes auto-Ubl modification, based on previous study (7). The UbaC RHD is proposed to be modified at its active-site cysteine by a persulfide sulfur (step 2). The source of this sulfur may be derived from cysteine by use of a cysteine desulfurase, such as the SufS described by Zafrilla et al. (57), that may cooperate with an RHD protein(s) in this process. UbaC binding to UbaA may occur prior to or after persulfide modification of the UbaC RHD. UbaC in this persulfide modified form may bind UbaA and directly attack the Ubl∼adenylate (step 4a) or the UbaA∼Ubl thioester (step 4b). We favor step 4a; however, either reaction (4a or b) would generate a Ubl acyl persulfide that would be susceptible to reductive cleavage resulting in the regeneration of UbaA and formation of thiocarboxylated Ubl (SAMP) (step 5) for use in the biosynthesis of MoCo and thiolation of tRNA (step 6). Unmodified UbaC may also bind the UbaA∼Ubl thioester (formed by reaction 3a) and transfer the Ubl to its active-site cysteine (step 3b). The resulting RHD∼Ubl thioester may undergo nucleophilic attack by the target protein to regenerate the UbaC RHD and form a Ubl protein conjugate (step 7). Note that MSRA is also required for certain Ubl ligations (11). Ubl modification would then target proteins (step 8) for transient inactivation and/or proteasome-mediated degradation (1, 3).
Sulfur relay pathways typically require persulfidic cysteines to be generated on a protein intermediate(s) even when thiocarboxylated Ubls are used to insert sulfur into the final biomolecule (30). The sulfur cascade is often initiated by a cysteine desulfurase that accepts sulfur from inorganic bisulfide, thiosulfate, or cysteine and transfers it to the cysteine residue of an acceptor protein to form new persulfide groups (30). For example, in E. coli, Moco biosynthesis relies upon the cysteine desulfurase IscS and the RHD protein YnjE to provide the sulfur for thiocarboxylation of the Ubl MoaD after its adenylation by the E1-like MoeB (3941). In some cases, the sulfur-accepting RHD is fused to the C terminus of an E1-like adenyltransferase domain, as observed for MOCS3, Cnx5 and Uba4 (32, 4244). In these eukaryotic E1-RHD fusions, the conserved catalytic cysteine at the RHD active-site loop can carry the persulfidic sulfur from a sulfur-donating cysteine desulfurase and relay it to a sulfur-accepting adenylated Ubl to form a Ubl thiocarboxylate (32, 4244). In yeast, the Uba4 RHD transfers the persulfidic sulfur to the Ubl Urm1 for 2-thiolation of tRNA, whereas human MOCS3 and plant Cnx5 are more flexible in delivery of the RHD persulfidic sulfur to distinct Ubls for the biosynthesis of Moco (human MOCS2A and plant Cnx7) and thiolated tRNA (human Urm1 and plant Urm11) (39, 43). Thus, RHDs can have dual functions in sulfur mobilization to form multiple types of biomolecules (i.e., Moco and thiolated tRNA).
Not all sulfur relay pathways use RHDs and Ubls. In E. coli, a series of persulfidic cysteines mediated by IscS, TusA, TusBCD, TusE, and MnmA relay sulfur to form 2-thiouridine at the wobble position of tRNA, with no evidence of an RHD or Ubl intermediate (45). Likewise, certain archaea from sulfide-rich environments apparently do not require cysteine desulfurases or RHDs to form sulfur-containing biomolecules. For example, Methanococcus maripaludis uses a ThiI (MMP1354) that lacks an RHD to relay sulfur to form 4-thiouridine (s4U) tRNA (thiolated at tRNA position 8) (46). This RHD-minus ThiI can use elemental S2− as a substrate to generate persulfide and disulfide intermediates on the cysteine residues of its PP-loop domain and form s4U tRNA independent of a cysteine desulfurase or an RHD (46). Instead of an RHD, a 3Fe-3S cluster formed on ThiI is required for this tRNA thiolation activity (47). While Methanococcus maripaludis does encode an RHD (MMP0900), this RHD associates with a PP-loop domain protein to synthesize 2-selenouridine tRNA (removing sulfur from tRNA and replacing it with selenium) (48). [3Fe-3S] clusters are also apparent in archaeal and eukaryal NcsA/Ncs6 homologs that bind Ubls and catalyze the final stage of wobble uridine tRNA thiolation (s2U) (48). Thus, even systems that use RHDs in the thiocarboxylation of Ubls may also depend upon [3Fe-3S] clusters for sulfur transfer to the final biomolecule.
Here the RHD UbaC was found to be required for functions associated with the formation of the sulfur-containing Moco and 2-thiolatied wobble uridine tRNA as well as Ubl modification in H. volcanii. Prior to analysis of the H. volcanii RHD mutant strains, genomic analysis provided limited insight into whether an RHD was required for archaeal E1 function. In SSNs, stand-alone RHDs, such as UbaB/C, clustered and shared edges with the RHDs fused to E1 domains; however, similar conclusions could be drawn for the RHDs fused to metallo-β-lactamase domains. While UbaB is in genomic synteny with UbaA, mutation of ubaB had no discernible effect on Ubl pathways (8), and, based on this study, UbaB does not share redundant function with UbaC. One possible role of UbaB could be to compete with UbaC for binding to UbaA and, thus, impair UbaC function. Here the tandem RHDs (HVO_0025 and HVO_0024) could not be correlated with cell function based on analysis of single-deletion strains. One possible explanation for this lack of phenotype could be that the tandem RHDs are redundant in function and catalyze a step analogous to that for Tum1, a tandem RHD required for thiocarboxylation of the Ubl Urm1 in yeast (23). We also note that H. volcanii, from moderate sulfide environments, has a close relative from sulfide/sulfur-rich springs, Haloferax sulfurifontis (49), that encodes 7 RHD homologs, including UbaC. Thus, RHDs do not appear to be rapidly lost by natural selection in sulfide-rich ecosystems. Whether RHDs are expressed and/or place archaea at a competitive advantage in sulfide-rich environments remains to be determined. One could envision that H. sulfurifontis is at a competitive advantage over archaea that lack RHDs when grown in an environment with fluctuating levels of sulfide.
Archaeal sampylation and eukaryotic urmylation function at the crossroad of Ubl sulfur relay and protein conjugation and thus are thought to be molecular fossils of eukaryotic systems that function exclusively in Ub/Ubl protein modification. Both sampylation and urmylation function independent of canonical E2 Ub/Ubl-conjugating enzymes. The C-terminal RHD of the eukaryotic E1-like Uba4 is instead proposed to serve as an E2-like module in urmylation and as a sulfur donor in tRNA thiolation (24, 25). Here the stand-alone UbaC is found to associate with the E1 UbaA at the crossroad of Ubl sulfur relay and sampylation in the archaeon H. volcanii. Thus, noncanonical E2 modules are proposed to be expanded to include stand-alone archaeal RHDs that associate with E1-like enzymes for function in Ubl protein modification.
Not all sampylation pathways are dependent upon UbaC. While the E1-like UbaA is required for the formation of all of the Ubl conjugates detected in this study, UbaC was not. Beyond formation of the SAMP1-MoaE conjugate, UbaC was not required for the linkage of Ubl SAMP1 to protein targets. Likewise, when cells were treated with bortezomib or NaOCl, SAMP2-conjugate levels were substantially reduced in the ΔubaC mutant but not abolished. Detection of residual Ubl ligation products in the ΔubaC mutant suggests that a cryptic E2 analog(s) that mediates UbaC-independent Ubl ligation may exist. Alternatively, certain Ubl pathways may require only the E1 and MSRA, the latter of which is related by convergent evolution to an E3 ligase MSRB-fold substrate receptor and is needed for the Ubl modifications induced by DMSO and MetO but not proteasome inhibitor or NaOCl (11).



Biochemicals and analytical-grade inorganic chemicals were purchased from Fisher Scientific (Atlanta, GA), Bio-Rad (Hercules, CA), and Sigma-Aldrich (St. Louis, MO). Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). DNA polymerases and restriction enzymes were from New England Biolabs (Ipswich, MA). Hi-Lo DNA standards were from Minnesota Molecular, Inc. (Minneapolis, MN). Hybond-P polyvinylidene fluoride (PVDF) membranes for Western blots were from GE Healthcare.

Strains and culture conditions.

The genotypic profiles of bacterial and haloarchaeal strains used in this study are detailed in Table S1 in the supplemental material. Nutrient broth cultures were grown with rotary agitation at 200 rpm unless otherwise noted. Bacterial cultures were grown at 37°C in Luria-Bertani (LB) medium supplemented with ampicillin (Amp) (100 μg·ml−1) as needed. H. volcanii strains were grown at 42°C in ATCC 974 complex medium and yeast-peptone-Casamino Acids (YPC) (50). For H. volcanii strains carrying the expression plasmids, media were supplemented with novobiocin (Nov, 0.2 μg·ml−1). All strains were stored in 20% glycerol stocks at −80°C and freshly streaked for isolated colonies for analysis.

DNA isolation, PCRs and agarose gel electrophoresis.

DNA was separated by electrophoresis using 0.8% or 2% (wt/vol) agarose gels in 1× TAE electrophoresis buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5). Gels were stained with ethidium bromide at 0.5 μg · ml−1 and photographed with a Mini visionary imaging system (Fotodyne, Hartland, WI). Sizes of the DNA fragments were estimated using Hi-Lo DNA molecular weight markers (Minnesota Molecular, Inc., Minneapolis, MN). Plasmid DNA was isolated from E. coli strains using a QIAprep spin miniprep kit (Qiagen, Valencia, CA). PCRs were carried out using primers detailed in Table S2 in the supplemental material and Phusion and Taq polymerase for cloning and colony-based confirmatory tests, respectively. PCR products were purified by MinElute (Qiagen).

Cloning and site-directed mutagenesis.

Amino acid exchange of UbaC Cys64 (conserved at the RHD active-site loop) to Ser was performed by PCR-based site-directed mutagenesis (Stratagene, La Jolla, CA). UbaC–Strep-tag II (pJAM983; for H. volcanii expression) was used as a template to generate pJAM2705 (UbaC C64S–Strep-tag II).

Chromosomal knockout of ubaC and in trans complementation.

The open reading frame hvo_1947 (encoding the putative RHD protein, UbaC) was targeted for markerless deletion from the chromosome of H. volcanii H26 using the pyrE2-based “pop-in/pop-out” method as previously described (51) using suicide plasmid pJAM1900. Transformants were plated on Hv-minimal salts-Casamino Acids-agar medium (50), and direct colony PCR screening was used to check for pop-ins. Four pop-in colonies were pooled in 4 ml ATCC 974 with 5-fluoroorotic acid (5-FOA) (50 μg · ml−1) and plated on the same medium with agar. Null mutants were screened for the absence of 370-bp PCR product using primers that amplify 0.5 kb upstream/downstream flanking regions of hvo_1947 (ubaC). Plasmids pJAM983 (UbaC–Strep-tag II), pJAM2705 (UbaC C64S–Strep-tag II), pJAM2716 (UbaB–Strep-tag II), and pJAM202c (empty vector) were transformed into H. volcanii ΔubaC. A similar approach was used to generate the other H. volcanii mutant strains listed in Table S1.

DMSO respiration.

H. volcanii cells from freshly streaked ATCC 974 plates were grown overnight in 4 ml YPC medium to log phase (optical density at 600 nm [OD600] of 0.4 to 0.6) and subcultured to 4 ml fresh YPC medium using a 0.25% (vol/vol) inoculum. Cells at log phase (OD600 of 0.4 to 0.6) were used as an inoculum to fresh YPC medium with DMSO (to a final concentration of 100 mM) in screw-cap tubes (9 ml, 13 by 100 mm) from a starting OD600 of 0.02. Tubes were filled completely with medium and capped with phenolic caps. Cells were incubated at 42°C without agitation, and the cell density was monitored as OD600 using a Spectronic20+ spectrophotometer (Thermo Scientific, Madison, WI). To examine tungstate as a molybdate antagonist, the medium was supplemented with tungstate (10 mM) and/or molybdate (1 mM) as indicated.

DMSO reductase activity.

H. volcanii cells were cultured in triplicate in 50 ml YPC medium with DMSO (100 mM) for 36 h at 42°C with agitation at 200 rpm. Cells were harvested by centrifugation (6,000 × g, 4°C), resuspended in 15 ml high-salt Tris buffer (2 M NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5), and lysed by French press thrice (2,000 lb/in2). Whole-cell lysate was clarified by centrifugation (17,000 × g, 4°C) and filtration (0.2 μm), and the protein concentration of the soluble fraction was measured using a bicinchoninic acid (BCA)-based protein assay kit. DMSO reductase activity was monitored by A600 (15-s intervals for 3.5 min) with nitrogen as the headspace. Assay mixtures (4 ml) included cell lysate (1 to 1.5 mg protein) and 0.3 mM methyl viologen in buffer A. The mixture was titrated with fresh 20 mM sodium dithionite (Na2S2O4) in 20 mM sodium bicarbonate (NaHCO3) until an A600 of 1 to 1.2 units was reached prior to addition of 10 mM DMSO. One unit of enzyme activity is defined as 1 μmol substrate consumed per min at room temperature with an extinction coefficient A600 of 13.6 mM−1 cm−1 for methyl viologen. All assays were performed in biological triplicate, with the means ± standard deviations (SD) calculated.

Thermal and oxidative stress assays.

H. volcanii cells from freshly streaked ATCC 974 plates were grown overnight in 4 ml ATCC 974 medium to log phase (OD600 of 0.4 to 0.6) and subcultured to fresh 4 ml ATCC 974 medium using a 0.25% (vol/vol) inoculum. Cells at log phase (OD600 of 0.4 to 0.6) were serially diluted onto ATCC 974 plates and grown at 42°C and 50°C. Alternatively, the log-phase cells were exposed to NaOCl (20 mM) or a mock control for 10 to 60 min, serially diluted, spotted onto ATCC 974 plates, and then incubated for 3 days at 42°C.

In vivo sampylation.

Flag-SAMPs, alone or with MoaE–Strep-tag II, UbaC–Strep-tag II, UbaA–Strep-tag II, or variant proteins, were expressed in H. volcanii. Novobiocin (0.2 μg · ml−1) was included in the medium to maintain the expression plasmids. Cells were grown overnight in 4 ml ATCC 974 and subcultured to 4 ml fresh ATCC 974 with DMSO (100 mM), methionine sulfoxide (MetO) (25 mM), or a mock control using a 0.25% (vol/vol) inoculum. Cells were grown to stationary phase (OD600 of 2.0 to 3.0) with agitation (200 rpm) at 42°C and harvested by centrifugation. Alternatively, cultures were supplemented with a proteasome inhibitor (100 μM bortezomib) or NaOCl (20 mM) at log phase and incubated overnight at 42°C (200 rpm). Cells were harvested and analyzed for sampylation (Ubl modifications) by immunoblotting.

Pulldown assay.

H. volcanii H26-pJAM983, which expresses UbaC–Strep-tag II, and H26-pJAM986, which expresses UbaC (untagged), were used for the pulldown assays. Cells were grown to log phase (OD600 of 0.8) in 50 ml ATCC 974 medium with novobiocin (0.2 μg · ml−1) in 250-ml flasks at 42°C with agitation (200-rpm rotary shaking). Harvested cells were lysed in 20 ml high-salt Tris buffer using a French press, and whole-cell lysate was clarified by centrifugation (20,000 × g for 10 min, 4°C) and filtration (0.45 μm). The protein concentration was determined using a BCA protein assay kit (Pierce). Clarified cell extract (1.5 mg protein in 1 ml lysate) was incubated with 50 μl Streptactin-Sepharose resin slurry (GE Healthcare) for 1 h at 4°C with gentle rocking to allow binding. The resin was washed thrice with 1 ml high-salt buffer. UbaC–Strep-tag II and its associated protein partners were eluted from the resin by the addition of 100 μl Tris-salt buffer (50 mM Tris, 150 mM NaCl, pH 7.5) supplemented with d-desthiobiotin (5 mM). Eluted proteins were separated by reducing SDS-PAGE (12%) and examined for protein constituents by immunoblotting using anti-UbaA and anti-Strep-tag II antibodies.

Immunoblotting (Western blotting).

Cell pellets were resuspended and boiled for 20 min in 2× reducing SDS-PAGE loading buffer (62.5 mM Tris-Cl buffer at pH 6.8 with 2% [wt/vol] SDS, 25% [vol/vol] glycerol, 0.1 mg · ml−1 bromophenol blue with 5% [vol/vol] β-mercaptoethanol). Proteins were separated by 12% SDS-PAGE. Loading amounts were normalized to 0.065 OD600 unit per lane, with equal loading confirmed by staining parallel gels with Coomassie brilliant blue for total protein (with regions of the gels stained for total protein displayed alongside the immunoblots for ease of comparison). Proteins were electroblotted from the SDS-polyacrylamide gels onto Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare Bio-Sciences, Piscataway, NJ) for 3 h at 90 V. Flag-tagged proteins were detected using alkaline phosphatase-linked anti-Flag M2 monoclonal antibodies (Sigma). Unconjugated rabbit anti-Strep-tag II polyclonal antibodies (GenScript USA, Piscataway, NJ) were used to detect Strep-tag II-tagged proteins. Polyclonal antibodies raised in rabbit against UbaA (Cocalico Biologicals, Reamstown, PA), found to be specific for UbaA by comparison of parent and ΔubaA mutant strains (28), were used to detect UbaA. The secondary antibodies used with the rabbit polyclonal antibodies were alkaline phosphatase-linked goat anti-rabbit IgG(H+L) antibodies (SouthernBiotech, Birmingham, AL). CDP-Star (Applied Biosystems, Carlsbad, CA) was used as the substrate to detect the alkaline phosphatase-conjugated antibodies. Chemiluminescent products were visualized with X-ray film (Hyperfilm; GE Healthcare Bio-Sciences).

Bioinformatic analyses.

Protein domain architectures, amino acid positions of the domains, and IPR superfamilies were according to InterPro (52). Archaeal clusters of orthologous genes (arCOGs) were according to Makarova et al. (53). Sequence similarity networks (SSNs) were generated using the Enzyme Function Initiative Tools-Enzyme Similarity Tool (EIF-EST) according to Gerlt et al. (54). The amino acid sequences of the archaeal E1 homologs of the Ub-activating enzyme superfamily (IPR035985) were input as a FASTA file into EIF-EST; the SSN generated at an alignment score of 46 at 40% amino acid sequence identity with a minimum length of 150 amino acids (aa) was used to assess the relationships of these archaeal E1s. The amino acid sequences of the RHDs from the MOCS3/Uba4 E1s (human, yeast, and plant) and from arCOG02021 were extracted and input as a FASTA file into EIF-EST; the SSN with an alignment score of 13, which represents 36% amino acid sequence identity, was used for the RHD comparison. The 3D structures of UbaC and UbaB were modeled using Phyre2 (55) and compared to the X-ray crystal structure of the MOCS3 RHD (PDB no. 3I2V) using Chimera (56).


We thank Friedhelm Pfeiffer (Max Planck Institute of Biochemistry, Martinsried, Germany) for his helpful comments to improve the manuscript.
Funds for this project were awarded to J.A.M.-F. through the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE-FG02-05ER15650) to advance understanding of biomolecules related to energy metabolism and through the National Institutes of Health (NIH R01 GM57498) to examine archaeal Ubl systems that share ancient ties with ubiquitination.
N.L.H. and J.A.M.-F. conceived, designed, and performed the experiments and wrote the manuscript.

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Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 201Number 151 August 2019
eLocator: 10.1128/jb.00254-19
Editor: William W. Metcalf, University of Illinois at Urbana Champaign


Received: 8 April 2019
Accepted: 7 May 2019
Published online: 10 July 2019


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  1. archaea
  2. DMSO reductase
  3. halophiles
  4. molybdenum cofactor (Moco)
  5. posttranslational modification
  6. proteasome
  7. rhodanese
  8. sulfur
  9. tRNA thiolation
  10. ubiquitination



Nathaniel L. Hepowit
Department of Microbiology and Cell Science, Institute of Food and Agricultural Science, University of Florida, Gainesville, Florida, USA
Julie A. Maupin-Furlow
Department of Microbiology and Cell Science, Institute of Food and Agricultural Science, University of Florida, Gainesville, Florida, USA
Genetics Institute, University of Florida, Gainesville, Florida, USA


William W. Metcalf
University of Illinois at Urbana Champaign


Address correspondence to Julie A. Maupin-Furlow, [email protected].

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