Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Carbonic anhydrases fused to anion transporters of the SulP family: evidence for a novel type of bicarbonate transporter

The sulfate permease (SulP) family of secondary carriers (TC #2.A.53) includes functionally characterized members that are inorganic anion:H+ symporters and anion:anion antiporters. We here describe members of this family that are fused to non-transporter domains, a relatively rare occurrence in prokaryotes. One subfamily includes members that are either fused to or are encoded within operons that also encode homologues of carbonic anhydrases, suggesting that these carriers function to take up bicarbonate or carbonate. Within another subfamily, a SulP homologue is fused to rhodanese, a thiosulfate:cyanide sulfotransferase, suggesting that this carrier functions in sulfate uptake. Some homologues are encoded in operons that also encode putative Na+/H+ antiporters of the NhaD family (TC #2.A.62) or putative Na+:HCO3- symporters of the SBT family (TC #2.A.83). SulP homologues present in fungi and some bacteria are fused to cyclic AMP-binding domains and STAS domains that presumably function in regulation or targeting. Phylogenetic analyses reveal the relationships of these proteins and protein domains to each other and show that in some cases, but not in others, the hydrophilic domains/proteins have coevolved with the transporters.     

Characterization of an Escherichia coli sulfite oxidase homologue reveals the role of a conserved active site cysteine in assembly and function

We report the biochemical and biophysical characterization of YedYZ, a sulfite oxidase homologue from Escherichia coli. YedY is a soluble catalytic subunit with a twin arginine leader sequence for export to the periplasm by the Tat translocation system. YedY is the only molybdoenzyme so far isolated from E. coli with the Mo-MPT form of the molybdenum cofactor. The electron paramagnetic resonance (EPR) signal of the YedY molybdenum is similar to that of known Mo-MPT containing enzymes, with the exception that only the Mo(IV) --> Mo(V) transition is observed, with a midpoint potential of 132 mV. YedZ is a membrane-intrinsic cytochrome b with six putative transmembrane helices. The single heme b of YedZ has a midpoint potential of -8 mV, determined by EPR spectroscopy of YedZ-enriched membrane preparations. YedY does not associate strongly with YedZ on the cytoplasmic membrane. However, mutation of the YedY active site Cys102 to Ser results in very efficient targeting of YedY to YedZ in the membrane, demonstrating a clear role for YedZ as the membrane anchor for YedY. Together, YedYZ comprise a well-conserved bacterial heme-molybdoenzyme found in a variety of bacteria that can be assigned to the sulfite oxidase class of enzyme.     

The copper transporter (Ctr) family of Cu+ uptake systems

Copper (Cu(+)) transporters of the Ctr family are sequence diverse eukaryotic proteins that function by an unknown mechanism of action. We have conducted bioinformatic analyses of sequenced Ctr proteins. Multiple paralogues are found in single organisms, and these may be either closely or distantly related to each other. Protein phylogeny generally correlates with organismal source and protein size with proteins of each cluster being derived from a specific eukaryotic kingdom and exhibiting characteristic domain arrangements. Some homologues exhibit repeats of the basic 3 TMS unit. Regions of conserved hydrophobicity and amphipathicity suggest functional roles, particularly for putative TMSs 2 and 3 which possess a nearly fully conserved M X(3) M motif in putative TMS2. We propose that this motif comprises the transmembrane Cu(+)-binding site in oligomeric channels that take up Cu(+) by a passive, membrane potential-dependent mechanism.     

Two Families of Mechanosensitive Channel Proteins

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Sequence analyses of cyanobacterial bicarbonate transporters and their homologues

The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA. SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs). These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product. A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin. Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters. The tree topology suggests that SbtA family members display multiple transport functions.     

Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans

PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.     

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members.     

Voltage-gated H+ channels associated with human phagocyte superoxide-generating NADPH oxidases: sequence comparisons,structural predictions,and phylogenetic analyses

The N-terminal domain of the human phagocyte flavocytochrome b558 NADPH oxidase, gp91phox, is believed to be a heme-containing voltage-gated H+ channel. The authors have conducted structural, sequence and phylogenetic analyses of the putative transmembrane channel/heme-binding domains of all homologous proteins in the NCBI GenBank database as of May 2001, as well as of the full-length proteins. Fifty-six homologues were identified, including 26 from animals, 19 from plants, seven from yeast, one from a slime mould and three from bacteria. Six well-defined sub-families were revealed by phylogenetic tree construction, two consisting of animal proteins, two of plant proteins, and one each of yeast and bacterial homologues, with the slime mould protein clustering loosely with one of the animal clusters. Signature sequences for the entire family as well as for the sub-families were determined. Most proteins have six putative TMSs, four of which may comprise the heme-binding H+ channel. The hydrophobic and amphipathic characteristics of each of the putative alpha-helical transmembrane segments were defined, and conserved residues that may be involved in heme binding, channel formation, and/or conformational changes were identified. The analyses lead to the suggestion that the oxidase domain became associated with the channel/heme-binding domain to form a single polypeptide chain early in evolutionary history, before eukaryotes diverged from prokaryotes, and that genetic transmission to present day organisms occurred primarily by vertical descent.     

Voltage-gated H + channels associated with human phagocyte superoxide-generating NADPH oxidases: Sequence comparisons,structural predictions,and phylogenetic analyses

The N-terminal domain of the human phagocyte flavocytochrome b 558 NADPH oxidase, gp91 phox, is believed to be a heme-containing voltage-gated H + channel. The authors have conducted structural, sequence and phylogenetic analyses of the putative transmembrane channel/heme-binding domains of all homologous proteins in the NCBI GenBank database as of May 2001, as well as of the full-length proteins. Fifty-six homologues were identified, including 26 from animals, 19 from plants, seven from yeast, one from a slime mould and three from bacteria. Six well-defined sub-families were revealed by phylogenetic tree construction, two consisting of animal proteins, two of plant proteins, and one each of yeast and bacterial homologues, with the slime mould protein clustering loosely with one of the animal clusters. Signature sequences for the entire family as well as for the sub-families were determined. Most proteins have six putative TMSs, four of which may comprise the heme-binding H + channel. The hydrophobic and amphipathic characteristics of each of the putative &#102 -helical transmembrane segments were defined, and conserved residues that may be involved in heme binding, channel formation, and/or conformational changes were identified. The analyses lead to the suggestion that the oxidase domain became associated with the channel/heme-binding domain to form a single polypeptide chain early in evolutionary history, before eukaryotes diverged from prokaryotes, and that genetic transmission to present day organisms occurred primarily by vertical descent.     

Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.     

Origin,diversification and substrate specificity in the family of NCS1/FUR transporters

NCS1 proteins are H⁺/Na⁺ symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub‐families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward‐facing selective gate. Our evolutionary and structure‐function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel‐like gates might contribute to substrate specificity.     

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.     

The five near-iron transporter (NEAT) domain anthrax hemophore, IsdX2, scavenges heme from hemoglobin and transfers heme to the surface protein IsdC

Pathogenic bacteria require iron to replicate inside mammalian hosts. Recent studies indicate that heme acquisition in Gram-positive bacteria is mediated by proteins containing one or more near-iron transporter (NEAT) domains. Bacillus anthracis is a spore-forming, Gram-positive pathogen and the causative agent of anthrax disease. The rapid, extensive, and efficient replication of B. anthracis in host tissues makes this pathogen an excellent model organism for the study of bacterial heme acquisition. B. anthracis secretes two NEAT hemophores, IsdX1 and IsdX2. IsdX1 contains a single NEAT domain, whereas IsdX2 has five, a novel property among hemophores. To understand the functional significance of harboring multiple, non-identical NEAT domains, we purified each individual NEAT domain of IsdX2 as a GST fusion and analyzed the specific function of each domain as it relates to heme acquisition and transport. NEAT domains 1, 3, 4, and 5 all bind heme, with domain 5 having the highest affinity. All NEATs associate with hemoglobin, but only NEAT1 and -5 can extract heme from hemoglobin, seemingly by a specific and active process. NEAT1, -3, and -4 transfer heme to IsdC, a cell wall-anchored anthrax NEAT protein. These results indicate that IsdX2 has all the features required to acquire heme from the host and transport heme to the bacterial cell wall. Additionally, these results suggest that IsdX2 may accelerate iron import rates by acting as a "heme sponge" that enhances B. anthracis replication in iron-starved environments.     

Evolution of the Ferric Reductase Domain (FRD) Superfamily: Modularity,Functional Diversification,and Signature Motifs

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.