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Microbial phenol oxidases and peroxidases mediate biogeochemical processes in soils, including microbial acquisition of carbon and nitrogen, lignin degradation, carbon mineralization and sequestration, and dissolved organic carbon export. Measuring oxidative enzyme activities in soils is more problematic than assaying hydrolytic enzyme activities because of the non-specific, free radical nature of the reactions and complex interactions between enzymes, assay substrates, and the soil matrix. We compared three substrates commonly used to assay phenol oxidase and peroxidase in soil: pyrogallol (PYGL, 1,2,3-trihydroxybenzene), l-DOPA (l-3,4-dihydroxyphenylalanine), and ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). We measured substrate oxidation in three soils across a pH gradient from 3.0 to 10.0 to determine the pH optimum for each substrate. In addition, we compared activities across 17 soils using the three substrates. In general, activities on the substrates followed the trend PYGL>. l-DOPA>ABTS and were inversely related to substrate redox potential. PYGL and ABTS were not suitable substrates at pH>5, and ABTS oxidation often declined with addition of peroxide to the assay. Absolute and relative oxidation rates varied widely among substrates in relation to soil type and assay pH. We also tested whether autoclaved or combusted soils could be used as negative controls for the influence of abiotic factors (e.g., soil mineralogy) on oxidative activity. However, neither autoclaving nor combustion produced reliable negative controls because substrate oxidation still occurred; in some cases, these treatments enhanced substrate oxidation rates. For broad scale studies, we recommend that investigators use all three substrates to assess soil oxidation potentials. For focused studies, we recommend evaluating substrates before choosing a single option, and we recommend assays at both the soil pH and a reference pH (e.g., pH 5.0) to determine the effect of assay pH on oxidase activity. These recommendations should contribute to greater comparability of oxidase potential activities across studies. © 2013 Elsevier Ltd.

Wildfire disturbance propagation along fluvial networks remains poorly understood. We use incident, atmospheric, and water-quality data from the largest wildfire in New Mexico's history to quantify how this gigafire affected surface runoff processes and mobilized wildfire disturbances into fluvial networks after burning 1382 km 2 . Surface runoff post-fire increased compared to pre-fire conditions, and precipitation events that are frequently observed in the affected watershed (<2-year recurrence) and fell during the post-fire first rainy season resulted in uncorrelated, less frequently observed runoff events (10-year recurrence). Besides these shifts in runoff generation, the magnitude and fluctuation of daily water quality parameters and relevant ecosystem processes also shifted over multiple months, even at sites located >160 km downstream of the burn perimeter. Our findings emphasize the need to incorporate spatially resolved longitudinal sampling designs into wildfire water quality research and highlight the spatiotemporal co-dependency among atmospheric, terrestrial, and aquatic processes in defining the net outcome of wildfire disturbance propagation along impacted fluvial networks., (© 2024. The Author(s).)

Extremophiles exist among all three domains of life; however, physiological mechanisms for surviving harsh environmental conditions differ among Bacteria, Archaea and Eukarya. Consequently, we expect that domain-specific variation of diversity and community assembly patterns exist along environmental gradients in extreme environments. We investigated inter-domain community compositional differences along a high-elevation salinity gradient in the McMurdo Dry Valleys, Antarctica. Conductivity for 24 soil samples collected along the gradient ranged widely from 50 to 8355 µS cm -1 . Taxonomic richness varied among domains, with a total of 359 bacterial, 2 archaeal, 56 fungal, and 69 non-fungal eukaryotic operational taxonomic units (OTUs). Richness for bacteria, archaea, fungi, and non-fungal eukaryotes declined with increasing conductivity (all P < 0.05). Principal coordinate ordination analysis (PCoA) revealed significant (ANOSIM R = 0.97) groupings of low/high salinity bacterial OTUs, while OTUs from other domains were not significantly clustered. Bacterial beta diversity was unimodally distributed along the gradient and had a nested structure driven by species losses, whereas in fungi and non-fungal eukaryotes beta diversity declined monotonically without strong evidence of nestedness. Thus, while increased salinity acts as a stressor in all domains, the mechanisms driving community assembly along the gradient differ substantially between the domains., (© 2022. The Author(s), under exclusive licence to Springer Japan KK, part of Springer Nature.)

Urban surface runoff from storms impacts the water quality dynamics of downstream ecosystems. While these effects are well-documented in mesic regions, they are not well constrained for arid watersheds, which sustain longer dry periods, receive intense but short-lived storms, and where stormwater drainage networks are generally isolated from sewage systems. We used a network of high-frequency in situ water quality sensors located along the Middle Rio Grande to determine surface runoff origins during storms and track rapid changes in physical, chemical, and biological components of water quality. Specific conductivity (SpCond) patterns were a reliable indicator of source, distinguishing between runoff events originating primarily in urban (SpCond sags) or non-urban (SpCond spikes) catchments. Urban events were characterized by high fluorescent dissolved organic matter (fDOM), low dissolved oxygen (including short-lived hypoxia <2 mg/L), smaller increases in turbidity and varied pH response. In contrast, non-urban events showed large turbidity spikes, smaller dissolved oxygen sags, and consistent pH sags. Principal component analysis distinguished urban and non-urban events by dividing physical and biogeochemical water quality parameters, and modeling of DO along the same reach demonstrated consistently higher oxygen demand for an urban event compared to a non-urban event. Based on our analysis, urban runoff poses more potential ecological harm, while non-urban runoff poses a larger problem for drinking water treatment. The comparison of our results to other reports of urban stormwater quality suggest that water quality responses to storm events in urban landscapes are consistent across a range of regional climates., Competing Interests: Declaration of competing interest The authors do not declare any conflict of interest in submitting this manuscript for publication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The distribution of organisms in an environment is neither uniform nor random but is instead spatially patterned. The factors that control this patterning are complex and the underlying mechanisms are poorly understood. Soil microbes are critical to ecosystem function but exhibit highly complex distributions and community dynamics due in large part to the scale-dependent effects of environmental heterogeneity. To better understand the impact of environmental heterogeneity on the distribution of soil microbes, we sequenced the 16S rRNA gene from bacterial communities in the microbe-dominated polar desert ecosystem of the McMurdo Dry Valleys (MDV), Antarctica. Significant differences in key edaphic variables and alpha diversity were observed among the three lake basins of the Taylor Valley (Kruskal-Wallis; pH: χ 2 = 68.89, P < 0.001, conductivity: χ 2 = 35.03, P < 0.001, observed species: χ 2 = 7.98, P = 0.019 and inverse Simpson: χ 2 = 18.52, P < 0.001) and each basin supported distinctive microbial communities (ANOSIM R = 0.466, P = 0.001, random forest ratio of 14.1). However, relationships between community structure and edaphic characteristics were highly variable and contextual, ranging in magnitude and direction across regional, basin, and local scales. Correlations among edaphic factors (pH and soil conductivity) and the relative abundance of specific phyla were most pronounced along local environmental gradients in the Lake Fryxell basin where Acidobacteria, Bacteroidetes, and Proteobacteria declined while Deinococcus-Thermus and Gemmatimonadetes increased with soil conductivity (all P < 0.1). Species richness was most strongly related to the soil conductivity gradient present within this study system. We suggest that the relative importance of pH versus soil conductivity in structuring microbial communities is related to the length of edaphic gradients and the spatial scale of sampling. These results highlight the importance of conducting studies over large ranges of key environmental gradients and across multiple spatial scales to assess the influence of environmental heterogeneity on the composition and diversity of microbial communities.

Microbial consortia dominate glacial meltwater streams from polar regions, including the McMurdo Dry Valleys (MDV), where they thrive under physiologically stressful conditions. In this study, we examined microbial mat types and sediments found in 12 hydrologically diverse streams to describe the community diversity and composition within and across sites. Sequencing of the 16S rRNA gene from 129 samples revealed ∼24 000 operational taxonomic units (<97% DNA similarity), making streams the most biodiverse habitat in the MDV. Principal coordinate analyses revealed significant but weak clustering by mat type across all streams (ANOSIM R-statistic = 0.28) but stronger clustering within streams (ANOSIM R-statistic from 0.28 to 0.94). Significant relationships (P < 0.05) were found between bacterial diversity and mat ash-free dry mass, suggesting that diversity is related to the hydrologic regimes of the various streams, which are predictive of mat biomass. However, correlations between stream chemistry and community members were weak, possibly reflecting the importance of internal processes and hydrologic conditions. Collectively, these results suggest that localized conditions dictate bacterial community composition of the same mat types and sediments from different streams, and while MDV streams are hotspots of biodiversity in an otherwise depauperate landscape, controls on community structure are complex and site specific., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Closing nutrient loops in terrestrial and aquatic ecosystems is integral to achieve resource security in the food-energy-water (FEW) nexus. We performed multiyear (2005-2008), monthly sampling of instream dissolved inorganic nutrient concentrations (NH4-N, NO3-N, soluble reactive phosphorus-SRP) along a ∼ 300-km arid-land river (Rio Grande, NM) and generated nutrient budgets to investigate how the net source/sink behavior of wastewater and irrigated agriculture can be holistically managed to improve water quality and close nutrient loops. Treated wastewater on average contributed over 90% of the instream dissolved inorganic nutrients (101 kg/day NH4-N, 1097 kg/day NO3-N, 656 kg/day SRP). During growing seasons, the irrigation network downstream of wastewater outfalls retained on average 37% of NO3-N and 45% of SRP inputs, with maximum retention exceeding 60% and 80% of NO3-N and SRP inputs, respectively. Accurate quantification of NH4-N retention was hindered by low loading and high variability. Nutrient retention in the irrigation network and instream processes together limited downstream export during growing seasons, with total retention of 33-99% of NO3-N inputs and 45-99% of SRP inputs. From our synoptic analysis, we identify trade-offs associated with wastewater reuse for agriculture within the scope of the FEW nexus and propose strategies for closing nutrient loops in arid-land rivers.

The soils of the McMurdo Dry Valleys, Antarctica are an extreme polar desert, inhabited exclusively by microscopic taxa. This region is on the threshold of anticipated climate change, with glacial melt, permafrost thaw, and the melting of massive buried ice increasing liquid water availability and mobilizing soil nutrients. Experimental water and organic matter (OM) amendments were applied to investigate how these climate change effects may impact the soil communities. To identify active taxa and their functions, total community RNA transcripts were sequenced and annotated, and amended soils were compared with unamended control soils using differential abundance and expression analyses. Overall, taxonomic diversity declined with amendments of water and OM. The domain Bacteria increased with both amendments while Eukaryota declined from 38% of all taxa in control soils to 8 and 11% in water and OM amended soils, respectively. Among bacterial phyla, Actinobacteria (59%) dominated water-amended soils and Firmicutes (45%) dominated OM amended soils. Three bacterial phyla (Actinobacteria, Proteobacteria, and Firmicutes) were primarily responsible for the observed positive functional responses, while eukaryotic taxa experienced the majority (27 of 34) of significant transcript losses. These results indicated that as climate changes in this region, a replacement of endemic taxa adapted to dry, oligotrophic conditions by generalist, copiotrophic taxa is likely.

Microbial mats are abundant in many alpine and polar aquatic ecosystems. With warmer temperatures, new hydrologic pathways are developing in these regions and increasing dissolved nutrient fluxes. In the McMurdo Dry Valleys, thermokarsting may release both nutrients and sediment, and has the potential to influence mats in glacial meltwater streams. To test the role of nutrient inputs on community structure, we created nutrient diffusing substrata (NDS) with agar enriched in N, P and N + P, with controls, and deployed them into two Dry Valley streams. We found N amendments (N and N + P) to have greater chlorophyll-a concentrations, total algal biovolume, more fine filamentous cyanobacteria and a higher proportion of live diatoms than other treatments. Furthermore, N treatments were substantially elevated in Bacteroidetes and the small diatom, Fistulifera pelliculosa. On the other hand, species richness was almost double in P and N + P treatments over others, and coccoid green algae and Proteobacteria were more abundant in both streams. Collectively, these data suggest that nutrients have the potential to stimulate growth and alter community structure in glacial meltwater stream microbial mats, and the recent erosion of permafrost and accelerated glacial melt will likely impact resident biota in polar lotic systems here and elsewhere., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The causes of biodiversity patterns are controversial and elusive due to complex environmental variation, covarying changes in communities, and lack of baseline and null theories to differentiate straightforward causes from more complex mechanisms. To address these limitations, we developed general diversity theory integrating metabolic principles with niche-based community assembly. We evaluated this theory by investigating patterns in the diversity and distribution of soil bacteria taxa across four orders of magnitude variation in spatial scale on an Antarctic mountainside in low complexity, highly oligotrophic soils. Our theory predicts that lower temperatures should reduce taxon niche widths along environmental gradients due to decreasing growth rates, and the changing niche widths should lead to contrasting α- and β-diversity patterns. In accord with the predictions, α-diversity, niche widths and occupancies decreased while β-diversity increased with increasing elevation and decreasing temperature. The theory also successfully predicts a hump-shaped relationship between α-diversity and pH and a negative relationship between α-diversity and salinity. Thus, a few simple principles explained systematic microbial diversity variation along multiple gradients. Such general theory can be used to disentangle baseline effects from more complex effects of temperature and other variables on biodiversity patterns in a variety of ecosystems and organisms., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Soil microbial communities of the McMurdo Dry Valleys, Antarctica (MDV) contain representatives from at least fourteen bacterial phyla. However, given low rates of microbial activity, it is unclear whether this richness represents functioning rather than dormant members of the community. We used stable isotope probing (SIP) with (18) O-water to determine if microbial populations grow in MDV soils. Changes in the microbial community were characterized in soils amended with H2 (18) O and H2 (18) O-organic matter. Sequencing the 16S rRNA genes of the heavy and light fractions of the bacterial community DNA shows that DNA of microbial populations was labeled with (18) O-water, indicating these micro-organisms grew in the MDV soils. Significant differences existed in the community composition of the heavy and light fractions of the H2 (18) O and H2 (18) O-organic matter amended samples (Anosim P < 0.05 of weighted Unifrac distance). Control samples and the light DNA fraction of the H2 (18) O amended samples were dominated by representatives of the phyla Deinococcus-Thermus, Proteobacteria, Planctomyces, Gemmatimonadetes, Actinobacteria and Acidobacteria, whereas Proteobacteria were more prevalent in the heavy DNA fractions from the H2 (18) O-water and the H2 (18) O-water-organic matter treatments. Our results indicate that SIP with H2 (18) O can be used to distinguish active bacterial populations even in this low organic matter environment., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Edaphic factors such as pH, organic matter, and salinity are often the most significant drivers of diversity patterns in soil bacterial communities. Desert ecosystems in particular are model locations for examining such relationships as food web complexity is low and the soil environment is biogeochemically heterogeneous. Here, we present the findings from a 16S rRNA gene sequencing approach used to observe the differences in diversity and community composition among three divergent soil habitats of the McMurdo Dry Valleys, Antarctica. Results show that alpha diversity is significantly lowered in high pH soils, which contain higher proportions of the phyla Acidobacteria and Actinobacteria, while mesic soils with higher soil organic carbon (and ammonium) content contain high proportions of Nitrospira, a nitrite-oxidizing bacteria. Taxonomic community resolution also had a significant impact on our conclusions, as pH was the primary predictor of phylum-level diversity, while moisture was the most significant predictor of diversity at the genus level. Predictive power also increased with increasing taxonomic resolution, suggesting a potential increase in niche-based drivers of bacterial community composition at such levels., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Microbial communities in extreme environments often have low diversity and specialized physiologies suggesting a limited resistance to change. The McMurdo Dry Valleys (MDV) are a microbially dominated, extreme ecosystem currently undergoing climate change-induced disturbances, including the melting of massive buried ice, cutting through of permafrost by streams, and warming events. These processes are increasing moisture across the landscape, altering conditions for soil communities by mobilizing nutrients and salts and stimulating autotrophic carbon inputs to soils. The goal of this study was to determine the effects of resource addition (water/organic matter) on the composition and function of microbial communities in the MDV along a natural salinity gradient representing an additional gradient of stress in an already extreme environment. Soil respiration and the activity of carbon-acquiring extracellular enzymes increased significantly (P < 0.05) with the addition of resources at the low- and moderate-salinity sites but not the high-salinity site. The bacterial community composition was altered, with an increase in Proteobacteria and Firmicutes with water and organic matter additions at the low- and moderate-salinity sites and a near dominance of Firmicutes at the high-salinity site. Principal coordinate analyses of all samples using a phylogenetically informed distance matrix (UniFrac) demonstrated discrete clustering among sites (analysis of similarity [ANOSIM], P < 0.05 and R > 0.40) and among most treatments within sites. The results from this experimental work suggest that microbial communities in this environment will undergo rapid change in response to the altered resources resulting from climate change impacts occurring in this region.

Understanding controls over the distribution of soil bacteria is a fundamental step toward describing soil ecosystems, understanding their functional capabilities, and predicting their responses to environmental change. This study investigated the controls on the biomass, species richness, and community structure and composition of soil bacterial communities in the McMurdo Dry Valleys, Antarctica, at local and regional scales. The goals of the study were to describe the relationships between abiotic characteristics and soil bacteria in this unique, microbially dominated environment, and to test the scale dependence of these relationships in a low complexity ecosystem. Samples were collected from dry mineral soils associated with snow patches, which are a significant source of water in this desert environment, at six sites located in the major basins of the Taylor and Wright Valleys. Samples were analyzed for a suite of characteristics including soil moisture, pH, electrical conductivity, soil organic matter, major nutrients and ions, microbial biomass, 16 S rRNA gene richness, and bacterial community structure and composition. Snow patches created local biogeochemical gradients while inter-basin comparisons encompassed landscape scale gradients enabling comparisons of microbial controls at two distinct spatial scales. At the organic carbon rich, mesic, low elevation sites Acidobacteria and Actinobacteria were prevalent, while Firmicutes and Proteobacteria were dominant at the high elevation, low moisture and biomass sites. Microbial parameters were significantly related with soil water content and edaphic characteristics including soil pH, organic matter, and sulfate. However, the magnitude and even the direction of these relationships varied across basins and the application of mixed effects models revealed evidence of significant contextual effects at local and regional scales. The results highlight the importance of the geographic scale of sampling when determining the controls on soil microbial community characteristics.

The degradation of detrital organic matter and assimilation of carbon (C), nitrogen (N), and phosphorus (P) by heterotrophic microbial communities is mediated by enzymes released into the environment (ecoenzymes). For the attached microbial communities of soils and freshwater sediments, the activities of β-glucosidase, β-N-acetylglucosaminidase, leucine aminopeptidase, and phosphatase show consistent stoichiometric patterns. To determine whether similar constraints apply to planktonic communities, we assembled data from nine studies that include measurements of these enzyme activities along with microbial productivity. By normalizing enzyme activity to productivity, we directly compared the ecoenzymatic stoichiometry of aquatic biofilm and bacterioplankton communities. The relationships between β-glucosidase and α-glucosidase and β-glucosidase and β-N-acetylglucosaminidase were statistically indistinguishable for the two community types, while the relationships between β-glucosidase and phosphatase and β-glucosidase and leucine aminopeptidase significantly differed. For β-glucosidase vs. phosphatase, the differences in slope (biofilm 0.65, plankton 1.05) corresponded with differences in the mean elemental C:P ratio of microbial biomass (60 and 106, respectively). For β-glucosidase vs. leucine aminopeptidase, differences in slope (0.80 and 1.02) did not correspond to differences in the mean elemental C:N of biomass (8.6 and 6.6). β-N-Acetylglucosaminidase activity in biofilms was significantly greater than that of plankton, suggesting that aminosaccharides were a relatively more important N source for biofilms, perhaps because fungi are more abundant. The slopes of β-glucosidase vs. (β-N-acetylglucosaminidase + leucine aminopeptidase) regressions (biofilm 1.07, plankton 0.94) corresponded more closely to the estimated difference in mean biomass C:N. Despite major differences in physical structure and trophic organization, biofilm and plankton communities have similar ecoenzymatic stoichiometry in relation to productivity and biomass composition. These relationships can be integrated into the stoichiometric and metabolic theories of ecology and used to analyze community metabolism in relation to resource constraints.

mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

Most RNA polymerase III transcripts are bound immediately after synthesis by an abundant nuclear phosphoprotein known as the La autoantigen. Experiments performed in the budding yeast Saccharomyces cerevisiae have revealed that binding of the La protein to tRNA precursors is required for the endonucleolytic maturation of the 3' terminus of many tRNAs. In the absence of this protein, the 3' ends of these tRNAs are trimmed by exonucleases (Yoo CJ, Wolin SL, 1997, Cell 89:393-402). Here we report the characterization of the La protein in the fission yeast Schizosaccharomyces pombe. As was described for budding yeast, S. pombe cells lacking the La protein are viable and exhibit alterations in the pathway of pre-tRNA maturation. Introduction of either the human, S. cerevisiae, or S. pombe La protein into these cells restores the detected pattern of tRNA processing intermediates to that of wild-type cells. By performing immunoprecipitations from cells that were metabolically labeled with 32P-orthophosphate, we demonstrate that the S. pombe and S. cerevisiae La proteins, like the human La protein, are phosphorylated in vivo. Thus, although the La protein is dispensable for growth in these yeasts, both the structure of the protein and its function in pre-tRNA maturation have been highly conserved throughout evolution.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.

In virtually all vertebrate cells, Ro RNPs consist of the 60-kDa Ro autoantigen bound to one of several small cytoplasmic RNA molecules known as Y RNAs. Because the 60-kDa Ro autoantigen is also found complexed with defective precursors of 5S rRNA in Xenopus oocytes, we have proposed that this protein functions in a quality control, or discard pathway, for 5S RNA biosynthesis (O'Brien CA, Wolin SL, 1994, Genes & Dev 8:2891-2903). The role of the Y RNAs in this pathway is unknown. To begin a genetic analysis of Ro RNP function, we have characterized these particles in the nematode Caenorhabditis elegans. The C. elegans Ro protein is 12 kDa larger than the vertebrate protein; the larger size is due in part to an N-terminal extension and to two insertions in the RNA recognition motif. In contrast to all previously described vertebrate species, the Ro protein appears bound to a single Y RNA in C. elegans. Similar to vertebrate Y RNAs, the C. elegans Y RNA can be folded to form a pyrimidine-rich internal loop and a long stem in which the 5' and 3' ends are base paired. Within the stem is a conserved bulged helix that is proposed to be the binding site of the Ro protein. Interestingly, although the human protein can bind the nematode Y RNA, the C. elegans protein does not bind human Y RNAs. This is the first description of Ro RNPs in an invertebrate species.

Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.

We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.