Functional Nonequivalence Research Articles

“If less is more, how you keeping score?” Outlines of a life cycle assessment method to assess sufficiency

It is increasingly clear that reaching environmental sustainability requires not only efficiency (reduced environmental impact per functionality) but also sufficiency measures (reduced environmental impact through reduced or changed functionality). Life cycle assessment (LCA) is a widely used tool to study environmental impacts related to consumption. However, because of the LCA convention of only comparing alternative products with equal functionality, it is currently inept as a method for assessing the environmental impacts of sufficiency measures. Against this background, this short paper aims to stimulate a discussion on how sufficiency measures can be assessed with LCA methodology. By analyzing the very few LCAs of explicit sufficiency measures in terms of the components of a functional unit (what function is provided, how much, for how long, and how well) features of a potential new branch of LCA methodology are outlined, called Sufficiency LCA. In Sufficiency LCA, product alternatives need to be similar enough so that the what component of the functional unit can be equal, while the other components, how much, how long, and how well, are allowed to be non-equal. Thus, a key feature of Sufficiency LCA concerns functional non-equivalence of compared product alternatives, which is not allowed or neglected in conventional LCA, but which could be allowed, acknowledged and quantified in Sufficiency LCA. Developing Sufficiency LCA could be critical considering that sufficiency measures are expected to be required, and that LCA is expected to be serviceable as decision-making support, in the transition toward environmental sustainability.

Regulation of enzymes with identical subunits on the example of Transketolase

The molecule of transketolase is a dimer with structurally and functionally identical subunits. Its active sites are located in the region of intersubunit contact, which has been shown also for other thiamine enzymes. This leads to the reciprocal influence of active sites in the binding of cofactors and during catalysis. In this review, it is shown that the functional non-equivalence of the active sites of transketolase from Saccharomyces cerevisiae is initially formed upon the binding of the first cofactor, a divalent cation, not thiamine diphosphate, as previously thought. An attempt was made to find the reason for the differences between catalytic measurements and crystallographic data on the possible part-of-the-sites reactivity of the functioning of transketolase. At the same time, the difference in the amplitudes of dichroic absorption during the binding of reversibly and irreversibly splitting substrates has nothing to do with the flip-flop mechanism. It was also shown that with an increase in the concentration of substrates, a sharp decrease in activity occurs, which is explained by a switch from the simultaneous binding of the substrate in two active sites to its alternate binding both in one-substrate and two-substrate reactions. This fact could also be the reason for the rejection of the flip-flop mechanism of catalysis by transketolase. The mechanism may be similar to human transketolase, which may have clinical application.

A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development.

Role of genomic imprinting in mammalian development

Non-mendelian inheritance refers to the group of phenomena and observations related to the inheritance of genetic information that cannot be merely explained by Mendel's laws of inheritance. Phenomenon including Genomic imprinting, X-chromosome Inactivation, Paramutations are some of the best studied examples of non-mendelian inheritance. Genomic imprinting is a process that reversibly marks one of the two homologous loci, chromosome or chromosomal sets during development, resulting in functional non-equivalence of gene expression. Genomic imprinting is known to occur in a few insect species, plants, and placental mammals. Over the years, studies on imprinted genes have contributed immensely to highlighting the role of epigenetic modifications and the epigenetic circuitry during gene expression and development. In this review, we discuss the phenomenon of genomic imprinting in mammals and the role it plays especially during fetoplacental growth and early development.

Chromosomal instability-induced senescence potentiates cell non-autonomous tumourigenic effects

Chromosomal instability (CIN), a high rate of chromosome loss or gain, is often associated with poor prognosis and drug resistance in cancers. Aneuploid, including near-polyploid, cells contain an abnormal number of chromosomes and exhibit CIN. The post-mitotic cell fates following generation of different degrees of chromosome mis-segregation and aneuploidy are unclear. Here we used aneuploidy inducers, nocodazole and reversine, to create different levels of aneuploidy. A higher extent of aneuploid and near-polyploid cells in a given population led to senescence. This was in contrast to cells with relatively lower levels of abnormal ploidy that continued to proliferate. Our findings revealed that senescence was accompanied by DNA damage and robust p53 activation. These senescent cells acquired the senescence-associated secretory phenotype (SASP). Depletion of p53 reduced the number of senescent cells with concomitant increase in cells undergoing DNA replication. Characterisation of these SASP factors demonstrated that they conferred paracrine pro-tumourigenic effects such as invasion, migration and angiogenesis both in vitro and in vivo. Finally, a correlation between increased aneuploidy and senescence was observed at the invasive front in breast carcinomas. Our findings demonstrate functional non-equivalence of discernable aneuploidies on tumourigenesis and suggest a cell non-autonomous mechanism by which aneuploidy-induced senescent cells and SASP can affect the tumour microenvironment to promote tumour progression.

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.

Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic "emergency release valve," is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E.coli I49 and the equivalent S.aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.

Aneuploid Cells Are Differentially Susceptible to Caspase-Mediated Death during Embryonic Cerebral Cortical Development

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

Functional nonequivalence of transketolase active centers

Transketolase (TK, EC 2.2.1.1), the key enzyme of the non-oxidative branch of pentose phosphate pathway of hydrocarbon transformation, plays an important role in a system of substrate rearrangement between pentose shunt and glycolysis, acting as a reversible link between the two metabolic pathways. In addition, it supplies precursors for biosyntheses of nucleotides, aromatic amino acids, and vitamins. In plants, the enzyme plays a central role in the Calvin cycle. TK catalyzes interconversion of sugar phosphates. Thiamine diphosphate (TDP) and bivalent cations serve as its cofactors. Being a typical TDP-dependent enzyme, TK is the least complex representative of this group of enzymes, and this accounts for its use as a model in studies of their structure and mechanism of action. TK is readily crystallized, this being the reason why the first crystal X-ray structure analysis of TDP-dependent enzymes was performed with a TK sample. Both the general structure of TK and the structures of its active centers have been studied in detail. In this article, we review experimental evidence of functional nonequivalence of the two active centers of TK, which are known to be identical by crystal X-ray structure analysis.

Restoration of Dlk1 and Rtl1 Is Necessary but Insufficient to Rescue Lethality in Intergenic Differentially Methylated Region (IG-DMR)-deficient Mice

In the Dlk1-Dio3 imprinted domain, an intergenic differentially methylated region (IG-DMR) regulates the parental allele-specific expression of imprinted genes. The maternally inherited deletion of IG-DMR (IG-DMR((-/+))) results in perinatal lethality because of the overexpression of paternally expressed genes and repression of maternally expressed noncoding RNAs (ncRNAs), including Gtl2. To better understand the possible contribution of paternally expressed genes to the lethality, we attempted to rescue the lethality of IG-DMR((-/+)) mutants by restoring the paternally expressed genes. Because the paternally inherited Gtl2 deletion (Gtl2((+/-))) induced a decrease in the expression of paternally expressed genes, we crossed female IG-DMR heterozygous mice and male Gtl2 heterozygous mutant mice. The resultant IG-DMR((-/+))/Gtl2((+/-)) double mutant mice had normal expression levels of paternally expressed genes, and none of them showed perinatal lethality; however, most mice showed postnatal lethality with decreased expression of the maternally expressed ncRNAs. Thus, we inferred that paternally expressed genes are necessary for perinatal survivability and that maternally expressed ncRNAs are involved in postnatal lethality.

Inhibition of transketolase by hexacyanoferrate(III)

The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.

HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes

BackgroundDuring the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.

The HECT Domain of TRIP12 Ubiquitinates Substrates of the Ubiquitin Fusion Degradation Pathway

The ubiquitin fusion degradation (UFD) pathway is a proteolytic system conserved in yeast and mammals in which an uncleavable ubiquitin moiety linked to the N terminus of a protein functions as a degradation signal of the fusion protein. Although key components of the UFD pathway in yeast have been identified, the E3 enzyme of the human UFD pathway has not been studied. In this work, we show that TRIP12 is the E3 enzyme of the human UFD pathway. Thus, TRIP12 catalyzes in vitro ubiquitination of UFD substrates in conjunction with E1, E2, and E4 enzymes. Knockdown of TRIP12 stabilizes not only artificial UFD substrates but a physiological substrate UBB+1. Moreover, TRIP12 knockdown reduces UBB+1-induced cell death in human neuroblastoma cells. Surprisingly, complementation of TRIP12 knockdown cells with the TRIP12 HECT domain mostly restores efficient degradation of UFD substrates, indicating that the TRIP12 HECT domain can act as the E3 enzyme for the UFD pathway in human cells. The TRIP12 HECT domain directs ubiquitination of UFD substrates in vitro and can be specifically cross-linked to the ubiquitin moiety of the substrates in vivo, suggesting that the TRIP12 HECT domain possesses a noncovalent ubiquitin-binding site. In addition, we demonstrate that UbDeltaGG, a mutant ubiquitin that cannot be conjugated to other proteins, is a substrate of the TRIP12 HECT domain both in vivo and in vitro, indicating that the C-terminal extension fused to the uncleavable ubiquitin is not required for substrate recognition in the UFD pathway. These results provide new insights into the mechanism of the mammalian UFD pathway and the functional nonequivalence of different HECT domains.

Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development

Genomic imprinting refers to the functional non-equivalence of parental genomes in mammals that results from the parent-of-origin allelic expression of a subset of genes. Parent-specific expression is dependent on the germ line acquisition of DNA methylation marks at imprinting control regions (ICRs), coordinated by the DNA-methyltransferase homolog DNMT3L. We discuss here how the gender-specific stages of DNMT3L expression may have influenced the various sexually dimorphic aspects of genomic imprinting: (1) the differential developmental timing of methylation establishment at paternally and maternally imprinted genes in each parental germ line, (2) the differential dependence on DNMT3L of parental methylation imprint establishment, (3) the unequal duration of paternal versus maternal methylation imprints during germ cell development, (4) the biased distribution of methylation-dependent ICRs towards the maternal genome, (5) the different genomic organization of paternal versus maternal ICRs, and finally (6) the overwhelming contribution of maternal germ line imprints to development compared to their paternal counterparts.

Direct Kinetic Evidence for Half-Of-The-Sites Reactivity in the E1 Component of the Human Pyruvate Dehydrogenase Multienzyme Complex through Alternating Sites Cofactor Activation

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

Lon is an ATP-dependent serine protease that degrades damaged and certain regulatory proteins in vivo. Lon exists as a homo-oligomer and represents one of the simplest ATP-dependent proteases because both the protease and ATPase domains are located within each monomeric subunit. Previous pre-steady-state kinetic studies revealed functional nonequivalency in the ATPase activity of the enzyme [Vineyard, D., et al. (2005) Biochemistry 44, 1671-1682]. Both a high- and low-affinity ATPase site has been previously reported for Lon [Menon, A. S., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 14921-14928]. Because of the differing affinities for ATP, we were able to monitor the activities of the sites separately and determine that they were noninteracting. The high-affinity sites hydrolyze ATP very slowly (k(obs) = 0.019 +/- 0.002 s(-1)), while the low-affinity sites hydrolyze ATP quickly at a rate of 17.2 +/- 0.09 s(-1), which is comparable to the previously observed burst rate. Although the high-affinity sites hydrolyze ATP slowly, they support multiple rounds of peptide hydrolysis, indicating that ATP and peptide hydrolysis are not stoichiometrically linked. However, ATP binding and hydrolysis at both the high- and low-affinity sites are necessary for optimal peptide cleavage and the stabilization of the conformational change associated with nucleotide binding.

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.

Genomic imprinting in plants: the epigenetic version of an Oedipus complex

Genomic imprinting is a mitotically stable epigenetic modification that results in the functional non-equivalency of both parental genomes following fertilization. In flowering plants, studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. In Arabidopsis, the Polycomb group protein MEDEA regulates seed development through the expression of the MADS-box gene PHERES1. Activation of the maternal MEDEA allele requires the function of DEMETER, a plant DNA glycosylase that also controls the transcriptional activity of the maternally inherited allele of the late-flowering gene FWA. Current studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. Our current understanding of parent-of-origin effects could represent a new form of an Oedipus complex in which flowering plants prefer to rely transcriptionally on their maternal rather than their paternal chromosomes to ensure normal initiation of seed development.

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.

The effects of introduced water hyacinth on habitat structure, invertebrate assemblages, and fish diets

The South American floating aquatic plant water hyacinth (Eichhornia crassipes) has a history of worldwide invasions, including a 1904 introduction into the Sacramento-San Joaquin Delta, California. The native pennywort (Hydrocotyle umbellata) occupies similar habitats in the Delta and is extensively used by resident invertebrates and fish. We sought to discover if an invader would be functionally equivalent to the native plant, by asking whether the encroadhing hyacinth modified the invertebrate assemblage structure and fish-invertebrate food web relative to pennywort. We sampled epiphytic, epibenthic, and benthic invertebrates, and plant canopy insects in patches of hyacinth and pennywort, and analyzed fish diets at three sites in the Delta during 1998. We also measured habitat structure (leaf density, root biomass, and surface area). In 1999, following control and absence of hyacinth, we again measured epiphytic invertebrates in pennywort. We found differences between hyacinth and pennywort in structure, associated invertebrates, and fish diets. Most measurements inferred functional non-equivalency between hyacinth and pennywort, although some functional equivalency and natural variation existed. Leaf and insect densities were significantly higher in pennywort and there were also significant differences in insect assemblage compositions. Hyacinth roots in the water column had significantly more surface area. Densities of epibenthic and benthic aquatic invertebrates were typically greater in pennywort and taxonomic compositions of aquatic invertebrate assemblages showed significant differences. Amphipods and isopods living epiphytically in the root masses were particularly abundant, including several newly discovered introduced species: the amphipodCrangonyx floridanus and the isopodsCaecidotea racovitai andAsellus hilgendorfii. The native amphipodHyalella azteca was more abundant in pennywort and heavily preyed upon by fish, while the non-indigenousC. floridanus was more abundant in hyacinth and not prevalent in fish diets. The introduction of hyacinth to the Delta has caused significant ecological alterations in the surrounding community, due to hyacinth being functionally different from native patches of pennywort.