Phylogenetic,Structural and Functional Characteristics of the Na-K-Cl Cotransporter Family

Summary Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane α-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggests that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom. Received: 27 July 1995/Revised: 8 November 1995     

Phylogenetic Characterization of the MIP Family of Transmembrane Channel Proteins

The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity for water and/or neutral solutes. We have identified 84 fully or partially sequenced members of this family, have multiply aligned over 50 representative, divergent, fully sequenced members, have used the resultant multiple alignment to derive current MIP family-specific signature sequences, and have constructed a phylogenetic tree. The tree reveals novel features relevant to the evolutionary history of this protein family. These features plus an evaluation of functional studies lead to the postulates: (i) that all current MIP family proteins derived from two divergent bacterial paralogues, one a glycerol facilitator, the other an aquaporin, and (ii) that most or all current members of the family have retained these or closely related physiological functions. Received: 19 April 1996/Revised: 3 June 1996     

Phylogenetic Analysis of Three Lipocalin-Like Proteins Present in the Milk of Trichosurus vulpecula (Phalangeridae, Marsupialia)

Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents. Received: 28 October 1996 / Accepted: 19 May 1997     

Relatedness and Phylogeny Within the Family of Periplasmic Chaperones Involved in the Assembly of Pili or Capsule-Like Structures of Gram-Negative Bacteria

The structure of a Salmonella enterica serovar typhi gene located within the fim gene cluster and encoding a putative periplasmic chaperone-like protein involved in the assembly of type 1 pili was determined. This gene, named fimC, has the ability to encode a 26-kDa polypeptide which is similar, at the sequence level, to the PapD periplasmic chaperonin mediating the assembly of P pili of Escherichia coli, as well as to other periplasmic chaperone-like proteins involved in the biogenesis of pili or capsule-like structures of various Gram-negative bacteria. A comprehensive search through the literature and sequence databases identified 31 (putative) bacterial proteins that can be included in this protein family on the basis of sequence similarity. Results of a multiple sequence comparison analysis showed that several residues, including most of those known to be critical in maintaining the three-dimensional structure of PapD, are either conserved or conservatively substituted in all these proteins, suggesting an overall similar folding for all of them. It was also evident that members of this family are clustered into different subfamilies according to structural and phyletic data. Received: 15 February 1996 / Accepted: 3 October 1996     

Isolation and Characterization of a Carbonic Anhydrase Homologue from the Zebrafish (Danio rerio)

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost. Received: 31 March 1996 / Accepted: 8 September 1996     

Characterization of Interrelated Sequence Motifs in Four Satellite DNAs and Their Distribution in the Genome of the Mollusc Donax trunculus

A family of four satellite DNAs has been characterized in the genome of the bivalve mollusc, Donax trunculus. All share HindIII sites, a similar monomer length of about 160 base pairs (bp), and the related oligonucleotide motifs GGTCA and GGGTTA, repeated six to 15 times within the repetitive units. The motif GGTCA is common to all members of the satellite family. It is present in three of them in both orientations, interspersed within nonrepetitive DNA sequences. The hexanucleotide GGGTTA appears to be the main building element of one of the satellites forming a prominent subrepeat structure in conjunction with the 5-bp motif. The former has been also found in perfect tandem repeats in a junction region adjacent to the proper satellite sequence. Southern analysis has revealed that (GGGTTA)_n and/or related sequences are abundant and widely distributed in the D. trunculus genome. The distribution observed is consistent with the concurrence of the scattering of short sequence motifs throughout the genome and the spread of longer DNA segments, with concomitant formation of satellite monomer repeats. Both kinds of dispersion may have contributed to the observed complex arrangement of the HindIII satellite DNA family in Donax. Received: 28 May 1996 / Accepted: 30 July 1996     

Signature Sequences in Diverse Proteins Provide Evidence of a Close Evolutionary Relationship Between the Deinococcus-Thermus Group and Cyanobacteria

A number of proteins have been identified that contain prominent sequence signatures that are uniquely shared by the members of the Deinococcus-Thermus genera and the cyanobacterial species but which are not found in any of the other eubacterial or archaebacterial homologs. The proteins containing such sequence signatures include (1) the DnaJ/Hsp40 family of proteins, (2) DNA polymerase I, (3) the protein synthesis elongation factor EF-Tu, and (4) the elongation factor EF-Ts. A strong affinity of the Deinococcus-Thermus species to cyanobacteria is also seen in the phylogenetic trees based on Hsp70 and DnaJ sequences. These results provide strong evidence of a close and specific evolutionary relationship between species belonging to these two eubacterial divisions. Received: 10 September 1997 / Accepted: 15 December 1997     

Sequence Analyses and Phylogenetic Characterization of the ZIP Family of Metal Ion Transport Proteins

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, α-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins. Received: 31 March 1997/Revised: 14 May 1998     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

A Protein Related to Eucaryal and Bacterial DNA-Motor Proteins in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the ``SMC family' involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called ``UvrA-related NTP-binding proteins superfamily' and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling. Received: 29 June 1996 / Accepted: 28 February 1997     

Duplication and divergence of the genes of the α-esterase cluster ofDrosophila melanogaster

The α-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle. Received: 18 January 1996 / Accepted: 12 March 1996     

The Molecular Clock Runs at Different Rates Among Closely Related Members of a Gene Family

The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D–binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to α-fetoprotein (AFP) and α-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin. Although individual family members display an approximate clock-like evolution, there are significant deviations—the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences. Received: 28 February 1995 / Accepted: 6 October 1997     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997     

A possible molecular ancestor for mollusk APGWamide,insect adipokinetic hormone,and crustacean red pigment concentrating hormone

Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar α helix–random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families. The APGWamide genes are larger than the AKH/RPCH genes. The sequence environment occupied by introns is similar in AKH/RPCH and APGWamide genes. Such similarities suggest that these peptide families might have been originated by gene rearrangements from a common ancestor having either an AKH/RPCH/APGWamide-like structure or both an AKH/RPCH-like and an APGWamide-like structures. In the former model, DNA fragments could have been gained when the ancestor evolved to mollusks and it could have lost nucleotides when the progression to mandibular arthropods took place. In the second model, AKH/RPCH-like structures could have been fused during evolution toward mandibular arthropods, whereas in mollusks they could have been lost with the possible amplification of the APGWamide-like structure. Loss of domains in exon 1 may have originated the signal peptide and the first codon of the active RPCH. In exon 2, loss of domains possibly determined the junctions of codons 2 to 5 with the loss of a APGWamide copy; exon 3 underwent fewer variations. The similarity of the mollusk APGWamide precursors is closer to that of the RPCH family than the insect AKH family, indicating an earlier evolutionary departure.     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

Of Worms and Men: An Evolutionary Perspective on the Fibroblast Growth Factor (FGF) and FGF Receptor Families

FGFs (fibroblast growth factors) play major roles in a number of developmental processes. Recent studies of several human disorders, and concurrent analysis of gene knock-out and properties of the corresponding recombinant proteins have shown that FGFs and their receptors are prominently involved in the development of the skeletal system in mammals. We have compared the sequences of the nine known mammalian FGFs, FGFs from other vertebrates, and three additional sequences that we extracted from existing databases: two human FGF sequences that we tentatively designated FGF10 and FGF11, and an FGF sequence from C?norhabditis elegans. Similarly, we have compared the sequences of the four FGF receptor paralogs found in chordates with four non-chordate FGF receptors, including one recently identified in C. elegans. The comparison of FGF and FGF receptor sequences in vertebrates and nonvertebrates shows that the FGF and FGF receptor families have evolved through phases of gene duplications, one of which may have coincided with the emergence of vertebrates, in relation with their new system of body scaffold. Received: 6 April 1996 / Accepted: 5 July 1996     

Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997