Bathymetric evolution of black corals through deep time.

Deep-sea lineages are generally thought to arise from shallow-water ancestors, but this hypothesis is based on a relatively small number of taxonomic groups. Anthozoans, which include corals and sea anemones, are significant contributors to the faunal diversity of the deep sea, but the timing and mechanisms of their invasion into this biome remain elusive. Here, we reconstruct a fully resolved, time-calibrated phylogeny of 83 species in the order Antipatharia (black coral) to investigate their bathymetric evolutionary history. Our reconstruction indicates that extant black coral lineages first diversified in continental slope depths (∼250-3000 m) during the early Silurian (∼437 millions of years ago (Ma)) and subsequently radiated into, and diversified within, both continental shelf (less than 250 m) and abyssal (greater than 3000 m) habitats. Ancestral state reconstruction analysis suggests that the appearance of morphological features that enhanced the ability of black corals to acquire nutrients coincided with their invasion of novel depths. Our findings have important conservation implications for anthozoan lineages, as the loss of 'source' slope lineages could threaten millions of years of evolutionary history and confound future invasion events, thereby warranting protection.

An Environmental DNA Primer for Microbial and Restoration Ecology.

Environmental DNA (eDNA) sequencing-DNA collected from the environment from living cells or shed DNA-was first developed for working with microbes and has greatly benefitted microbial ecologists for decades since. These tools have only become increasingly powerful with the advent of metabarcoding and metagenomics. Most new studies that examine diverse assemblages of bacteria, archaea, protists, fungi, and viruses lean heavily into eDNA using these newer technologies, as the necessary sequencing technology and bioinformatic tools have become increasingly affordable and user friendly. However, eDNA methods are rapidly evolving, and sometimes it can feel overwhelming to simply keep up with the basics. In this review, we provide a starting point for microbial ecologists who are new to DNA-based methods by detailing the eDNA methods that are most pertinent, including study design, sample collection and storage, selecting the right sequencing technology, lab protocols, equipment, and a few bioinformatic tools. Furthermore, we focus on how eDNA work can benefit restoration and what modifications are needed when working in this subfield.

Phylogenomics, Origin, and Diversification of Anthozoans (Phylum Cnidaria).

Anthozoan cnidarians (corals and sea anemones) include some of the world's most important foundation species, capable of building massive reef complexes that support entire ecosystems. Although previous molecular phylogenetic analyses have revealed widespread homoplasy of the morphological characters traditionally used to define orders and families of anthozoans, analyses using mitochondrial genes or rDNA have failed to resolve many key nodes in the phylogeny. With a fully resolved, time-calibrated phylogeny for 234 species constructed from hundreds of ultraconserved elements and exon loci, we explore the evolutionary origins of the major clades of Anthozoa and some of their salient morphological features. The phylogeny supports reciprocally monophyletic Hexacorallia and Octocorallia, with Ceriantharia as the earliest diverging hexacorals; two reciprocally monophyletic clades of Octocorallia; and monophyly of all hexacoral orders with the exception of the enigmatic sea anemone Relicanthus daphneae. Divergence dating analyses place Anthozoa in the Cryogenian to Tonian periods (648-894 Ma), older than has been suggested by previous studies. Ancestral state reconstructions indicate that the ancestral anthozoan was a solitary polyp that had bilateral symmetry and lacked a skeleton. Colonial growth forms and the ability to precipitate calcium carbonate evolved in the Ediacaran (578 Ma) and Cambrian (503 Ma) respectively; these hallmarks of reef-building species have subsequently arisen multiple times independently in different orders. Anthozoans formed associations with photosymbionts by the Devonian (383 Ma), and photosymbioses have been gained and lost repeatedly in all orders. Together, these results have profound implications for the interpretation of the Precambrian environment and the early evolution of metazoans.[Bilateral symmetry; coloniality; coral; early metazoans; exon capture; Hexacorallia; Octocorallia photosymbiosis; sea anemone; ultraconserved elements.].

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis.

The emission of cerium oxide nanoparticles (CeO2) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO2-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO2 (0.15 to 7mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28days after CeO2 (3.5mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO2-exposed rats at 28days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO2-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-ß or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO2 nanoparticle exposure.

A Global eDNA Comparison of Freshwater Bacterioplankton Assemblages Focusing on Large-River Floodplain Lakes of Brazil.

With its network of lotic and lentic habitats that shift during changes in seasonal connection, the tropical and subtropical large-river systems represent possibly the most dynamic of all aquatic environments. Pelagic water samples were collected from Brazilian floodplain lakes (total n = 58) in four flood-pulsed systems (Amazon [n = 21], Araguaia [n = 14], Paraná [n = 15], and Pantanal [n = 8]) in 2011-2012 and sequenced via 454 for bacterial environmental DNA using 16S amplicons; additional abiotic field and laboratory measurements were collected for the assayed lakes. We report here a global comparison of the bacterioplankton makeup of freshwater systems, focusing on a comparison of Brazilian lakes with similar freshwater systems across the globe. The results indicate a surprising similarity at higher taxonomic levels of the bacterioplankton in Brazilian freshwater with global sites. However, substantial novel diversity at the family level was also observed for the Brazilian freshwater systems. Brazilian freshwater bacterioplankton richness was relatively average globally. Ordination results indicate that Brazilian bacterioplankton composition is unique from other areas of the globe. Using Brazil-only ordinations, floodplain system differentiation most strongly correlated with dissolved oxygen, pH, and phosphate. Our data on Brazilian freshwater systems in combination with analysis of a collection of freshwater environmental samples from across the globe offers the first regional picture of bacterioplankton diversity in these important freshwater systems.

Effects of pulmonary exposure to chemically-distinct welding fumes on neuroendocrine markers of toxicity.

Exposure to welding fumes may result in disorders of the pulmonary, cardiovascular, and reproductive systems. Welders are also at a greater risk of developing symptoms similar to those seen in individuals with idiopathic Parkinson's disease. In welders, there are studies that suggest that alterations in circulating prolactin concentrations may be indicative of injury to the dopamine (DA) neurons in the substantia nigra. The goal of these studies was to use an established model of welding particulate exposure to mimic the effects of welding fume inhalation on reproductive functions. Since previous investigators suggested that changes in circulating prolactin may be an early marker of DA neuron injury, movement disorders, and reproductive dysfunction, prolactin, hypothalamic tyrosine hydroxylase (TH) levels (a marker of DA synthesis), and other measures of hypothalamic-pituitary-gonadal (HPG) function were measured after repetitive instillation of welding fume particulates generated by flux core arc-hard surfacing (FCA-HS), manual metal arc-hard surfacing (MMA-HS) or gas metal arc-mild steel (GMA-MS) welding, or manganese chloride (MnCl2). Exposure to welding fume particulate resulted in the accumulation of various metals in the pituitary and testes of rats, along with changes in hypothalamic TH and serum prolactin levels. Exposure to particulates with high concentrations of soluble manganese (Mn) appeared to exert the greatest influence on TH activity levels and serum prolactin concentrations. Thus, circulating prolactin levels may serve as a biomarker for welding fume/Mn-induced neurotoxicity. Other reproductive measures were collected, and these data were consistent with epidemiological findings that prolactin and testosterone may serve as biomarkers of welding particulate induced DA neuron and reproductive dysfunction.

A RNA-seq approach to identify putative toxins from acrorhagi in aggressive and non-aggressive Anthopleura elegantissima polyps.

BACKGROUND: The use of venom in intraspecific aggression is uncommon and venom-transmitting structures specifically used for intraspecific competition are found in few lineages of venomous taxa. Next-generation transcriptome sequencing allows robust characterization of venom diversity and exploration of functionally unique tissues. Using a tissue-specific RNA-seq approach, we investigate the venom composition and gene ontology diversity of acrorhagi, specialized structures used in intraspecific competition, in aggressive and non-aggressive polyps of the aggregating sea anemone Anthopleura elegantissima (Cnidaria: Anthozoa: Hexacorallia: Actiniaria: Actiniidae). RESULTS: Collectively, we generated approximately 450,000 transcripts from acrorhagi of aggressive and non-aggressive polyps. For both transcriptomes we identified 65 candidate sea anemone toxin genes, representing phospholipase A2s, cytolysins, neurotoxins, and acrorhagins. When compared to previously characterized sea anemone toxin assemblages, each transcriptome revealed greater within-species sequence divergence across all toxin types. The transcriptome of the aggressive polyp had a higher abundance of type II voltage gated potassium channel toxins/Kunitz-type protease inhibitors and type II acrorhagins. Using toxin-like proteins from other venomous taxa, we also identified 612 candidate toxin-like transcripts with signaling regions, potentially unidentified secretory toxin-like proteins. Among these, metallopeptidases and cysteine rich (CRISP) candidate transcripts were in high abundance. Furthermore, our gene ontology analyses identified a high prevalence of genes associated with "blood coagulation" and "positive regulation of apoptosis", as well as "nucleoside: sodium symporter activity" and "ion channel binding". The resulting assemblage of expressed genes may represent synergistic proteins associated with toxins or proteins related to the morphology and behavior exhibited by the aggressive polyp. CONCLUSION: We implement a multifaceted approach to investigate the assemblage of expressed genes specifically within acrorhagi, specialized structures used only for intraspecific competition. By combining differential expression, phylogenetic, and gene ontology analyses, we identify several candidate toxins and other potentially important proteins in acrorhagi of A. elegantissima. Although not all of the toxins identified are used in intraspecific competition, our analysis highlights some candidates that may play a vital role in intraspecific competition. Our findings provide a framework for further investigation into components of venom used exclusively for intraspecific competition in acrorhagi-bearing sea anemones and potentially other venomous animals.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

Complete mitochondrial genomes of the black corals Alternatipathesmirabilis Opresko & Molodtsova, 2021 and Parantipatheslarix (Esper, 1788) (Cnidaria, Anthozoa, Hexacorallia, Antipatharia, Schizopathidae).

We describe the complete mitogenomes of the black corals Alternatipathesmirabilis Opresko & Molodtsova, 2021 and Parantipatheslarix (Esper, 1790) (Cnidaria, Anthozoa, Hexacorallia, Antipatharia, Schizopathidae). The analysed specimens include the holotype of Alternatipathesmirabilis, collected from Derickson Seamount (North Pacific Ocean; Gulf of Alaska) at 4,685 m depth and a potential topotype of Parantipatheslarix, collected from Secca dei Candelieri (Mediterranean Sea; Tyrrhenian Sea; Salerno Gulf; Italy) at 131 m depth. We also assemble, annotate and make available nine additional black coral mitogenomes that were included in a recent phylogeny (Quattrini et al. 2023b), but not made easily accessible on GenBank. This is the first study to present and compare two mitogenomes from the same species of black coral (Stauropathesarctica (Lütken, 1871)) and, thus, place minimum boundaries on the expected level of intraspecific variation at the mitogenome level. We also compare interspecific variation at the mitogenome-level across five different specimens of Parantipathes Brook, 1889 (representing at least two different species) from the NE Atlantic and Mediterranean Sea.

The epidemiology of life-threatening complications associated with reproductive process in public hospitals in Argentina.

OBJECTIVE: To analyse life-threatening obstetric complications that occurred in public hospitals in Argentina. DESIGN: Multicentre collaborative cross-sectional study. SETTING: Twenty-five hospitals included in the Perinatal Network of Buenos Aires Metropolitan Area. POPULATION: Women giving birth in participating hospitals during a 1-year period. METHODS: All cases of severe maternal morbidity (SMM) and maternal mortality (MM) during pregnancy (including miscarriage and induced abortion), labour and puerperium were included. Data were collected prospectively. MAIN OUTCOME MEASURES: Identification criteria, main causes and incidence of SMM; case-fatality rates, morbidity-mortality index and effective intervention's use rate. RESULTS: A total of 552 women with life-threatening conditions were identified: 518 with SMM, 34 with MM. Identification criteria for SMM were case-management (48.9%), organ dysfunction (15.2%) and mixed criteria (35.9%). Incidence of SMM was 0.8% (95% confidence interval [95% CI] 0.73-0.87%) and hospital maternal death ratio was 52.3 per 100 000 live births (95% CI 35.5-69.1). Main causes of MM were abortion complications and puerperal sepsis; main causes of SMM were postpartum haemorrhage and hypertension. Overall case-fatality rate was 6.2% (95% CI 4.4-8.6): the highest due to sepsis (14.8%) and abortion complications (13.3%). Morbidity-mortality index was 15:1 (95% CI 7.5-30.8). Use rate of known effective interventions to prevent or treat main causes of MM and SMM was 52.3% (95% CI 46.9-57.7). CONCLUSIONS: This study describes the importance of life-threatening obstetric complications that took place in public hospitals with comprehensive obstetric care and the low utilisation of known effective interventions that may decrease rates of SMM and MM. It also provides arguments that justify the need to develop a surveillance system for SMM.

Pulmonary and cardiovascular responses of rats to inhalation of a commercial antimicrobial spray containing titanium dioxide nanoparticles.

Our laboratory has previously demonstrated that application of an antimicrobial spray product containing titanium dioxide (TiO(2)) generates an aerosol of titanium dioxide in the breathing zone of the applicator. The present report describes the design of an automated spray system and the characterization of the aerosol delivered to a whole body inhalation chamber. This system produced stable airborne levels of TiO(2) particles with a median count size diameter of 110 nm. Rats were exposed to 314 mg/m(3) min (low dose), 826 mg/m(3) min (medium dose), and 3638 mg/m(3) min (high dose) of TiO(2) under the following conditions: 2.62 mg/m(3) for 2 h, 1.72 mg/m(3) 4 h/day for 2 days, and 3.79 mg/m(3) 4 h/day for 4 days, respectively. Pulmonary (breathing rate, specific airway resistance, inflammation, and lung damage) and cardiovascular (the responsiveness of the tail artery to constrictor or dilatory agents) endpoints were monitored 24 h post-exposure. No significant pulmonary or cardiovascular changes were noted at low and middle dose levels. However, the high dose caused significant increases in breathing rate, pulmonary inflammation, and lung cell injury. Results suggest that occasional consumer use of this antimicrobial spray product should not be a hazard. However, extended exposure of workers routinely applying this product to surfaces should be avoided. During application, care should be taken to minimize exposure by working under well ventilated conditions and by employing respiratory protection as needed. It would be prudent to avoid exposure to children or those with pre-existing respiratory disease.

New genus and species of black coral from the SW Pacific and Antarctica (Cnidaria: Anthozoa: Antipatharia: Schizopathidae).

A new genus, Diplopathes, in the family Schizopathidae, and three new species are described from the Southwest Pacific and Antarctic region based on morphological data. The new genus superficially resembles Telopathes in being branched and having simple, bilateral pinnules, but differs in having strictly alternately arranged pinnules, and in having small polyps 4 mm or less in transverse diameter. Mitochondrial DNA placed Diplopathes and Telopathes in separate clades within the Schizopathidae, thus supporting the significance of seemingly subtle anatomical differences. The new species are: D. antarctica, with sparse branching, pinnules of up to 7 cm long, and polypar spines up to 0.045 mm tall; D. multipinnata, with dense branching, pinnules up to 3 cm long, and polypar spines up to 0.1 mm tall; and D. tuatoruensis, with very sparse branching, pinnules up to 10 cm long, and polypar spines up to 0.1 mm. Interestingly, the three new species do not form a monophyletic clade based on mitochondrial DNA. We propose and discuss two hypotheses to explain the results of the phylogenetic reconstruction, including that molecular and physical change are uncoupled or that we have uncovered another example of morphological convergence in unrelated species.

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (-2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Contrasting modes of mitochondrial genome evolution in sister taxa of wood-eating marine bivalves (Teredinidae and Xylophagaidae).

The bivalve families Teredinidae and Xylophagaidae include voracious consumers of wood in shallow and deep-water marine environments, respectively. The taxa are sister clades whose members consume wood as food with the aid of intracellular cellulolytic endosymbionts housed in their gills. This combination of adaptations is found in no other group of animals and was likely present in the common ancestor of both families. Despite these commonalities, the two families have followed dramatically different evolutionary paths with respect to anatomy, life history and distribution. Here we present 42 new mitochondrial genome sequences from Teredinidae and Xylophagaidae and show that distinct trajectories have also occurred in the evolution and organization of their mitochondrial genomes. Teredinidae display significantly greater rates of amino acid substitution but absolute conservation of protein-coding gene order, whereas Xylophagaidae display significantly less amino acid change but have undergone numerous and diverse changes in genome organization since their divergence from a common ancestor. As with many bivalves, these mitochondrial genomes encode two ribosomal RNAs, 12 protein coding genes, and 22 tRNAs; atp8 was not detected. We further show that their phylogeny, as inferred from amino acid sequences of 12 concatenated mitochondrial protein-coding genes, is largely congruent with those inferred from their nuclear genomes based on 18S and 28S ribosomal RNA sequences. Our results provide a robust phylogenetic framework to explore the tempo and mode of mitochondrial genome evolution and offer directions for future phylogenetic and taxonomic studies of wood-boring bivalves.

Description of two new genera and two new species of antipatharian corals in the family Aphanipathidae (Cnidaria: Anthozoa: Antipatharia).

Two new genera and two new species of black corals are recognized in the family Aphanipathidae. The new genus Anozopathes, with the species A. hawaiiensis sp. nov. and A. palauensis, sp. nov. is characterized by a sparsely and irregularly branched corallum with relatively long branches which can be straight, curved or crooked. The genus Aphanostichopathes, with the type species Cirripathes paucispina Brook, is characterized by an unbranched corallum with a long, curved stem with loose distal coils. Mitochondrial DNA data (nad5-IGR-nad1 for Anozopathes and cox3-cox1 for Aphanostichopathes) indicate that both taxa are related to the genera Aphanipathes, Phanopathes and Acanthopathes in the family Aphanipathidae, and morphologically they both share the characteristic of having spines with distinct conical tubercles. The two new species of Anozopathes are separated primarily by differences in colony growth form and in the size and shape of the skeletal spines. Species of Aphanostichopathes are separated primarily by differences in the size and shape of the spines and by size and density of the tubercles on the surface of the spines.

Morphological and molecular description of a new genus and species of black coral (Cnidaria: Anthozoa: Hexacorallia: Antipatharia: Antipathidae: Blastopathes) from Papua New Guinea.

Blastopathes medusa gen. nov., sp. nov., is described from Kimbe Bay, Papua New Guinea, based on morphological and molecular data. Blastopathes, assigned to the Antipathidae, is a large, mythology-inspiring black coral characterized by clusters of elongate stem-like branches that extend out at their base and then curve upward. Colonies are not pinnulate and contain single branches, which could represent new branch cluster formations. Morphological and molecular (mitochondrial DNA and targeted capture of nuclear loci) evidence supporting the establishment of a new genus is discussed. This is the first study to utilize the target capture of ultraconserved elements (UCEs) and exonic loci to elucidate phylogenetic relationships among black corals and to identify and place a new genus and species.

Ultra-gentle soft robotic fingers induce minimal transcriptomic response in a fragile marine animal.

Tessler et al. demonstrate that a 'soft' robot causes less stress to a jellyfish while handling compared to a traditional 'hard' robot.

Palaeoclimate ocean conditions shaped the evolution of corals and their skeletons through deep time.

Identifying how past environmental conditions shaped the evolution of corals and their skeletal traits provides a framework for predicting their persistence and that of their non-calcifying relatives under impending global warming and ocean acidification. Here we show that ocean geochemistry, particularly aragonite-calcite seas, drives patterns of morphological evolution in anthozoans (corals, sea anemones) by examining skeletal traits in the context of a robust, time-calibrated phylogeny. The lability of skeletal composition among octocorals suggests a greater ability to adapt to changes in ocean chemistry compared with the homogeneity of the aragonitic skeleton of scleractinian corals. Pulses of diversification in anthozoans follow mass extinctions and reef crises, with sea anemones and proteinaceous corals filling empty niches as tropical reef builders went extinct. Changing environmental conditions will likely diminish aragonitic reef-building scleractinians, but the evolutionary history of the Anthozoa suggests other groups will persist and diversify in their wake.

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme.

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K-ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.

Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.