Expression and localization of monocarboxylate transporters and sodium/proton exchangers in bovine rumen epithelium

Monocarboxylate-H+ cotransporters, such as monocarboxylate transporter (MCT) SLC16A, have been suggested to mediate transruminal fluxes of short-chain fatty acids, ketone bodies, and lactate. Using an RT-PCR approach, we demonstrate expression of MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping expressed sequence tag data allowed compilation of the complete open reading frames for MCT1 and MCT2. Immunohistochemical localization of MCT1 shows plasma membrane staining in cells of the stratum basale, with intense staining of the basal aspects of the cells. Immunostaining decreased in the cell layers toward the rumen lumen, with weak staining in the stratum spinsoum. Immunostaining in the stratum granulosum and stratum corneum was essentially negative. Since monocarboxylate transport will load the cytosol with acid, expression and location of Na+/H+ exchanger (NHE) family members within the rumen epithelium were determined. RT-PCR demonstrates expression of multiple NHE family members, including NHE1, NHE2, NHE3, and NHE8. In contrast to MCT1, immunostaining showed that NHE1 was predominantly localized to the stratum granulosum, with a progressive decrease toward the stratum basale. NHE2 immunostaining was observed mainly at an intracellular location in the stratum basale, stratum spinosum, and stratum granulosum. Given the anatomic localization of MCT1, NHE1, and NHE2, the mechanism of transruminal short-chain fatty acid, ketone body, and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum, with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.     

Emerging roles of alkali cation/proton exchangers in organellar homeostasis

The regulated movement of monovalent cations such as H(+), Li(+), Na(+) and K(+) across biological membranes influences a myriad of cellular processes and is fundamental to all living organisms. This is accomplished by a multiplicity of ion channels, pumps and transporters. Our insight into their molecular, cellular and physiological diversity has increased greatly in the past few years with the advent of genome sequencing, genetic manipulation and sophisticated imaging techniques. One of the revelations from these studies is the emergence of novel alkali cation/protons exchangers that are present in endomembranes, where they function to regulate not only intraorganellar pH but also vesicular biogenesis, trafficking and other aspects of cellular homeostasis.     

Changing ideas about eukaryotic origins

The origin of eukaryotic cells is one of the most fascinating challenges in biology, and has inspired decades of controversy and debate. Recent work has led to major upheavals in our understanding of eukaryotic origins and has catalysed new debates about the roles of endosymbiosis and gene flow across the tree of life. Improved methods of phylogenetic analysis support scenarios in which the host cell for the mitochondrial endosymbiont was a member of the Archaea, and new technologies for sampling the genomes of environmental prokaryotes have allowed investigators to home in on closer relatives of founding symbiotic partners. The inference and interpretation of phylogenetic trees from genomic data remains at the centre of many of these debates, and there is increasing recognition that trees built using inadequate methods can prove misleading, whether describing the relationship of eukaryotes to other cells or the root of the universal tree. New statistical approaches show promise for addressing these questions but they come with their own computational challenges. The papers in this theme issue discuss recent progress on the origin of eukaryotic cells and genomes, highlight some of the ongoing debates, and suggest possible routes to future progress.     

Evolutionary origins of bacterial bioluminescence

In bacteria, most genes required for the bioluminescence phenotype are contained in lux operons. Sequence alignments of several lux gene products show the existence of at least two groups of paralogous products. The alpha- and beta-subunits of bacterial luciferase and the non-fluorescent flavoprotein are paralogous, and two antennae proteins (lumazine protein and yellow fluorescence protein) are paralogous with riboflavin synthetase. Models describing the evolution of these paralogous proteins are suggested, as well as a postulate for the identity of the gene encoding a protobioluminescent luciferase.     

Evolutionary relationships of eukaryotic kingdoms

The evolutionary relationships of four eukaryotic kingdoms—Animalia, Plantae, Fungi, and Protista—remain unclear. In particular, statistical support for the closeness of animals to fungi rather than to plants is lacking, and a preferred branching order of these and other eukaryotic lineages is still controversial even though molecular sequences from diverse eukaryotic taxa have been analyzed. We report a statistical analysis of 214 sequences of nuclear small-subunit ribosomal RNA (srRNA) gene undertaken to clarify these evolutionary relationships. We have considered the variability of substitution rates and the nonindependence of nucleotide substitution across sites in the srRNA gene in testing alternative hypotheses regarding the branching patterns of eukaryote phylogeny. We find that the rates of evolution among sites in the srRNA sequences vary substantially and are approximately gamma distributed with size and shape parameter equal to 0.76. Our results suggest that (1) the animals and true fungi are indeed closer to each other than to any other crown group in the eukaryote tree, (2) red algae are the closest relatives of animals, true fungi, and green plants, and (3) the heterokonts and alveolates probably evolved prior to the divergence of red algae and animal-fungus-green-plant lineages. Furthermore, our analyses indicate that the branching order of the eukaryotic lineages that diverged prior to the evolution of alveolates may be generally difficult to resolve with the srRNA sequence data.     

The origins of eukaryotic gene structure

Most of the phenotypic diversity that we perceive in the natural world is directly attributable to the peculiar structure of the eukaryotic gene, which harbors numerous embellishments relative to the situation in prokaryotes. The most profound changes include introns that must be spliced out of precursor mRNAs, transcribed but untranslated leader and trailer sequences (untranslated regions), modular regulatory elements that drive patterns of gene expression, and expansive intergenic regions that harbor additional diffuse control mechanisms. Explaining the origins of these features is difficult because they each impose an intrinsic disadvantage by increasing the genic mutation rate to defective alleles. To address these issues, a general hypothesis for the emergence of eukaryotic gene structure is provided here. Extensive information on absolute population sizes, recombination rates, and mutation rates strongly supports the view that eukaryotes have reduced genetic effective population sizes relative to prokaryotes, with especially extreme reductions being the rule in multicellular lineages. The resultant increase in the power of random genetic drift appears to be sufficient to overwhelm the weak mutational disadvantages associated with most novel aspects of the eukaryotic gene, supporting the idea that most such changes are simple outcomes of semi-neutral processes rather than direct products of natural selection. However, by establishing an essentially permanent change in the population-genetic environment permissive to the genome-wide repatterning of gene structure, the eukaryotic condition also promoted a reliable resource from which natural selection could secondarily build novel forms of organismal complexity. Under this hypothesis, arguments based on molecular, cellular, and/or physiological constraints are insufficient to explain the disparities in gene, genomic, and phenotypic complexity between prokaryotes and eukaryotes.     

Evolutionary origins of the mammary gland

Because the mammary gland has no known homologue among the extant reptiles, attempts to reconstruct its evolution must focus on evidence from living mammals. Of the numerous structures that have been hypothesized to have given rise to the mammary gland, only three remain as plausible progenitors: sebaceous glands, eccrine glands and apocrine glands. Ancestral mammary glands are usually assumed to have produced a copious watery secretion like that of human eccrine sweat glands. However, in terms of anatomy, physiology, development and topographical distribution, mammary glands are more similar to apocrine and sebaceous glands than to typical eccrine glands. Nevertheless, each of the three populations of cutaneous glands exhibit specializations unlikely to be primitive for the mammary gland. The mammary gland either predated full differentiation of mammalian cutaneous glands or, more probably, evolved as a neomorphic mosaic that combined the properties of apocrine and sebaceous glands. Consequently, ancestral, prototypic lacteal glands may have had the capacity to synthesize and secrete small amounts of organic substances, as do sebaceous and apocrine glands of living mammals.     

Evolutionary origins of mechanosensitive ion channels

According to the recent revision, the universal phylogenetic tree is composed of three domains: Eukarya (eukaryotes), Bacteria (eubacteria) and Archaea (archaebacteria). Mechanosensitive (MS) ion channels have been documented in cells belonging to all three domains suggesting their very early appearance during evolution of life on Earth. The channels show great diversity in conductance, selectivity and voltage dependence, while sharing the property of being gated by mechanical stimuli exerted on cell membranes. In prokaryotes, MS channels were first documented in Bacteria followed by their discovery in Archaea. The finding of MS channels in archaeal cells helped to recognize and establish the evolutionary relationship between bacterial and archaeal MS channels and to show that this relationship extends to eukaryotic Fungi (Schizosaccharomyces pombe) and Plants (Arabidopsis thaliana). Similar to their bacterial and archaeal homologues, MS channels in eukaryotic cell-walled Fungi and Plants may serve in protecting the cellular plasma membrane from excessive dilation and rupture that may occur during osmotic stress. This review summarizes briefly some of the recent developments in the MS channel research field that may ultimately lead to elucidation of the biophysical and evolutionary principles underlying the mechanosensory transduction in living cells.     

Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana

Background  

All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing.     

Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements

Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.     

Evolutionary origins and diversification of proteobacterial mutualists

Mutualistic bacteria infect most eukaryotic species in nearly every biome. Nonetheless, two dilemmas remain unresolved about bacterial–eukaryote mutualisms: how do mutualist phenotypes originate in bacterial lineages and to what degree do mutualists traits drive or hinder bacterial diversification? Here, we reconstructed the phylogeny of the hyperdiverse phylum Proteobacteria to investigate the origins and evolutionary diversification of mutualistic bacterial phenotypes. Our ancestral state reconstructions (ASRs) inferred a range of 34–39 independent origins of mutualist phenotypes in Proteobacteria, revealing the surprising frequency with which host-beneficial traits have evolved in this phylum. We found proteobacterial mutualists to be more often derived from parasitic than from free-living ancestors, consistent with the untested paradigm that bacterial mutualists most often evolve from pathogens. Strikingly, we inferred that mutualists exhibit a negative net diversification rate (speciation minus extinction), which suggests that mutualism evolves primarily via transitions from other states rather than diversification within mutualist taxa. Moreover, our ASRs infer that proteobacterial mutualist lineages exhibit a paucity of reversals to parasitism or to free-living status. This evolutionary conservatism of mutualism is contrary to long-standing theory, which predicts that selection should often favour mutants in microbial mutualist populations that exploit or abandon more slowly evolving eukaryotic hosts.     

Evolutionary origins of metabolic compartmentalization in eukaryotes

Many genes in eukaryotes are acquisitions from the free-living antecedents of chloroplasts and mitochondria. But there is no evolutionary ‘homing device’ that automatically directs the protein product of a transferred gene back to the organelle of its provenance. Instead, the products of genes acquired from endosymbionts can explore all targeting possibilities within the cell. They often replace pre-existing host genes, or even whole pathways. But the transfer of an enzymatic pathway from one compartment to another poses severe problems: over evolutionary time, the enzymes of the pathway acquire their targeting signals for the new compartment individually, not in unison. Until the whole pathway is established in the new compartment, newly routed individual enzymes are useless, and their genes will be lost through mutation. Here it is suggested that pathways attain novel compartmentation variants via a ‘minor mistargeting’ mechanism. If protein targeting in eukaryotic cells possesses enough imperfection such that small amounts of entire pathways continuously enter novel compartments, selectable units of biochemical function would exist in new compartments, and the genes could become selected. Dual-targeting of proteins is indeed very common within eukaryotic cells, suggesting that targeting variation required for this minor mistargeting mechanism to operate exists in nature.     

The activities of eukaryotic replication origins in chromatin

DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.     

Cation/proton antiport systems in escherichia coli: properties of the sodium/proton antiporter.

The sodium/proton antiport system of Escherichia coli has been characterized by the effect of Na⁺ on the pH gradient established by respiration in everted membrane vesicles. The system has equal affinity for Na⁺ and Li⁺. Between pH 7 and 9 dissipation of Δψ, membrane potential, has no effect on the affinity for Na⁺ but decreases the V of the antiport reaction. Uptake of ²²Na⁺ by everted membrane vesicles was observed using flow dialysis.     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.