bfb,a Novel ENU-Induced blebs Mutant Resulting from a Missense Mutation in Fras1

Fras1 is an extracellular matrix associated protein with essential roles in adhesion of epithelia and mesenchyme during early embryonic development. The adhesive function of Fras1 is achieved through interaction with a group of related proteins, Frem 1–3, and a cytoplasmic adaptor protein Grip1. Mutation of each of these proteins results in characteristic epithelial blistering and have therefore become known as “blebs” proteins. Human Fraser syndrome presents with a similar phenotype and the blebs mice have been instrumental in identification of the genetic basis of Fraser syndrome. We have identified a new ENU-induced blebs allele resulting from a novel missense mutation in Fras1. The resulting mouse strain, blood filled blisters (bfb), presents with a classic blebs phenotype but does not exhibit embryonic lethality typical of other blebs mutants and in addition, we report novel palate and sternal defects. Analysis of the bfb phenotype confirms the presence of epithelial-mesenchymal adhesion defects but also supports the emerging role of blebs proteins in regulating signalling during organogenesis. The bfb strain provides new opportunities to investigate the role of Fras1 in development.     

PDBsum1 : A standalone program for generating PDBsum analyses

PDBsum1 is a standalone set of programs to perform the same structural analyses as provided by the PDBsum web server (https://www.ebi.ac.uk/pdbsum). The server has pages for every entry in the Protein Data Bank (PDB) and can also process user‐uploaded PDB files, returning a password‐protected set of pages that are retained for around 3 months. The standalone version described here allows for in‐house processing and indefinite retention of the results. All data files and images are pre‐generated, rather than on‐the‐fly as in the web version, so can be easily accessed. The program runs on Linux, Windows, and mac operating systems and is freely available for academic use at https://www.ebi.ac.uk/thornton-srv/software/PDBsum1.     

ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H⁺ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H⁺ symporter, with K_m 0.45±0.07 mM and V_max 0.57±0.02 mmol h⁻¹ (gdw) ⁻¹. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H⁺ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.     

177Lu-DOTA-HH1, a Novel Anti-CD37 Radio-Immunoconjugate: A Study of Toxicity in Nude Mice

Background

CD37 is an internalizing B-cell antigen expressed on Non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia cells (CLL). The anti-CD37 monoclonal antibody HH1 was conjugated to the bifunctional chelator p-SCN-Bn-DOTA and labelled with the beta-particle emitting radionuclide ¹⁷⁷Lu creating the radio-immunoconjugate (RIC) ¹⁷⁷Lu-DOTA-HH1 (¹⁷⁷Lu-HH1, trade name Betalutin). The present toxicity study was performed prior to initiation of clinical studieswith ¹⁷⁷Lu-HH1.

Methodology/Principal Findings

Nude mice with or without tumor xenografts were treated with 50 to 1000 MBq/kg ¹⁷⁷Lu- HH1 and followed for clinical signs of toxicity up to ten months. Acute, life threatening bone marrow toxicity was observed in animals receiving 800 and 1000 MBq/kg ¹⁷⁷Lu-HH1. Significant changes in serum concentrations of liver enzymes were evident for treatment with 1000 MBq/kg ¹⁷⁷Lu-HH1. Lymphoid depletion, liver necrosis and atrophy, and interstitial cell hyperplasia of the ovaries were also observed for mice in this dose group.

Conclusions/Significance

¹⁷⁷Lu-DOTA-HH1 was well tolerated at dosages about 10 times above those considered relevant for radioimmunotherapy in patients with B-cell derived malignancies.The toxicity profile was as expected for RICs. Our experimental results have paved the way for clinical evaluation of ¹⁷⁷Lu-HH1 in NHL patients.     

Functional Consequences of a Novel Variant of PCSK1

Background

Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.

Methodology/Principal Findings

A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.

Conclusion/Significance

Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.     

Metabolism of d-Glycero-d-Manno-Heptitol, Volemitol, in Polyanthus. Discovery of a Novel Ketose Reductase

Volemitol (d-glycero-d-manno-heptitol, α-sedoheptitol) is an unusual seven-carbon sugar alcohol that fulfills several important physiological functions in certain species of the genus Primula. Using the horticultural hybrid polyanthus (Primula × polyantha) as our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with concentrations of up to 50 mg/g fresh weight in source leaves (about 25% of the dry weight), followed by sedoheptulose (d-altro-2-heptulose, 36 mg/g fresh weight), and sucrose (4 mg/g fresh weight). Volemitol was shown by the ethylenediaminetetraacetate-exudation technique to be a prominent phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of the phloem sap carbohydrates, surpassed only by sucrose (63%). Preliminary ¹⁴CO₂ pulse-chase radiolabeling experiments showed that volemitol was a major photosynthetic product, preceded by the structurally related ketose sedoheptulose. Finally, we present evidence for a novel NADPH-dependent ketose reductase, tentatively called sedoheptulose reductase, in volemitol-containing Primula species, and propose it as responsible for the biosynthesis of volemitol in planta. Using enzyme extracts from polyanthus leaves, we determined that sedoheptulose reductase has a pH optimum between 7.0 and 8.0, a very high substrate specificity, and displays saturable concentration dependence for both sedoheptulose (apparent K_m = 21 mm) and NADPH (apparent K_m = 0.4 mm). Our results suggest that volemitol is important in certain Primula species as a photosynthetic product, phloem translocate, and storage carbohydrate.Alditols (sugar alcohols or acyclic polyols) may be chemically described as reduction products of aldose or ketose sugars. The most prevalent plant alditols are the hexitols sorbitol, mannitol, and galactitol. However, as many as 17 different alditols occur naturally in higher plants (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996). The lesser-known alditols are often restricted in their occurrence but still fulfill important functions in those plants where they do occur. Volemitol (Fig. ​(Fig.1) 1) is a good example of a less common but important alditol. This seven-carbon sugar alcohol seems to be confined to certain sections of the genus Primula, so much so that it has been suggested as a useful chemotaxonomical marker (Kremer, 1978). Very little is known about the physiology and metabolism of volemitol in primulas, except that it was an early photosynthetic product in cowslip (Primula veris) and oxslip (Primula elatior) (Kremer, 1978). Figure 1Fischer projections of volemitol and its four structurally related seven-carbon sugars. Nomenclature follows that of Collins (1987); trivial names are underlined.The physiological roles of alditols are manifold and largely resemble those of disaccharides and oligosaccharides. They include photosynthetic assimilation, translocation and storage of carbon, and reducing power, as well as protection against different types of stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the pentitol ribitol have been established in higher plants. They generally use NADPH as a hydrogen donor and aldose phosphate as a hydrogen acceptor, in concert with the corresponding phosphatases. One exception might be galactitol, which was suggested to be formed directly from unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar alditols are thought to be phloem-mobile (Lewis, 1984), this has only been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora and Madore, 1993).To expand our knowledge of alditol metabolism in higher plants beyond that of hexitols, we studied the carbohydrate metabolism of polyanthus (Primula × polyantha). This popular horticultural hybrid of primrose (Primula vulgaris), oxlip, and cowslip (Mabberley, 1997) was chosen because preliminary experiments showed that its volemitol content is very high, similar to that of the wild-type species, and because it may be easily grown both outdoors and indoors.We give a general overview on volemitol metabolism in polyanthus with special emphasis on the role of volemitol in plant development and phloem transport. We also report on a novel enzyme, a NADPH-dependent ketose reductase, which forms volemitol by the reduction of sedoheptulose.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

Bmovo-1 Regulates Ovary Size in the Silkworm,Bombyx mori

The regulation of antagonistic OVO isoforms is critical for germline formation and differentiation in Drosophila. However, little is known about genes related to ovary development. In this study, we cloned the Bombyx mori ovo gene and investigated its four alternatively spliced isoforms. BmOVO-1, BmOVO-2 and BmOVO-3 all had four C2H2 type zinc fingers, but differed at the N-terminal ends, while BmOVO-4 had a single zinc finger. Bmovo-1, Bmovo-2 and Bmovo-4 showed the highest levels of mRNA in ovaries, while Bmovo-3 was primarily expressed in testes. The mRNA expression pattern suggested that Bmovo expression was related to ovary development. RNAi and transgenic techniques were used to analyze the biological function of Bmovo. The results showed that when the Bmovo gene was downregulated, oviposition number decreased. Upregulation of Bmovo-1 in the gonads of transgenic silkworms increased oviposition number and elevated the trehalose contents of hemolymph and ovaries. We concluded that Bmovo-1 was involved in protein synthesis, contributing to the development of ovaries and oviposition number in silkworms.     

A New Enpp1 allele, Enpp1ttw-Ham,Identified in an ICR Closed Colony

We recently have reported on a novel ankylosis gene that is closely linked to theEnpp1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene onchromosome 10. Here, we have discovered novel mutant mice in a Jcl:ICR closed colony withankylosis in the toes of the forelimbs at about 3 weeks of age. The mutant mice exhibitedrigidity in almost all joints, including the vertebral column, which increased with age.These mice also showed hypogrowth with age after 16 weeks due to a loss of visceral fat,which may have been caused by poor nutrition. Histological examination and soft X-rayimaging demonstrated the ectopic ossification of various joints in the mutant mice. Inparticular, increased calcium deposits were observed in the joints of the toes, the carpalbones and the vertebral column. We sequenced all exons and exon/intron boundaries ofEnpp1 in the normal and mutant mice, and identified a G-to-Tsubstitution (c.259+1G>T) in the 5′ splice donor site of intron 2 in theEnpp1 gene of the mutant mice. This substitution led to the skipping ofexon 2 (73 bp), which generated a stop codon at position 354 bp (amino acid 62) of thecDNA (p.V63Xfs). Nucleotide pyrophosphohydrolase (NPPH) activity of ENPP1 in the mutantmice was also decreased, suggesting that Enpp1 gene function is disruptedin this novel mutant. The mutant mice reported in this study will be a valuable animalmodel for future studies of human osteochondral diseases and malnutrition.     

Saccharomyces cerevisiae Duo1p and Dam1p,Novel Proteins Involved in Mitotic Spindle Function

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.     

Medusa: A Novel Gene Drive System for Confined Suppression of Insect Populations

Gene drive systems provide novel opportunities for insect population suppression by driving genes that confer a fitness cost into pest or disease vector populations; however regulatory issues arise when genes are capable of spreading across international borders. Gene drive systems displaying threshold properties provide a solution since they can be confined to local populations and eliminated through dilution with wild-types. We propose a novel, threshold-dependent gene drive system, Medusa, capable of inducing a local and reversible population crash. Medusa consists of four components - two on the X chromosome, and two on the Y chromosome. A maternally-expressed, X-linked toxin and a zygotically-expressed, Y-linked antidote results in suppression of the female population and selection for the presence of the transgene-bearing Y because only male offspring of Medusa-bearing females are protected from the effects of the toxin. At the same time, the combination of a zygotically-expressed, Y-linked toxin and a zygotically-expressed, X-linked antidote selects for the transgene-bearing X in the presence of the transgene-bearing Y. Together these chromosomes create a balanced lethal system that spreads while selecting against females when present above a certain threshold frequency. Simple population dynamic models show that an all-male release of Medusa males, carried out over six generations, is expected to induce a population crash within 12 generations for modest release sizes on the order of the wild population size. Re-invasion of non-transgenic insects into a suppressed population can result in a population rebound; however this can be prevented through regular releases of modest numbers of Medusa males. Finally, we outline how Medusa could be engineered with currently available molecular tools.     

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background

Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.

Methodology/Principal Findings

We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.

Conclusions/Significance

PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.     

Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast: THE ENDOSOME/PREVACUOLAR COMPARTMENT IS A TARGET FOR SALT TOXICITY*

Mutants of the plant cation/H⁺ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by ³¹P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H⁺ antiporter.The Arabidopsis thaliana vacuolar alkali cation transporter AtNHX1 has been shown to increase salt tolerance in transgenic plants of several species (1). In Saccharomyces cerevisiae, its ortholog (ScNHX1) is mainly localized in late endosomes, where it is thought to contribute to vacuole biogenesis by regulating pH and vesicle volume (2). ScNHX1 itself has a role in halotolerance. Deletion of ScNHX1 confers salt sensitivity and diminishes Na⁺ compartmentalization, albeit indirectly, since the unrelated VNX1 exchanger accounts for most of the cation/H⁺ antiport activity in the tonoplast of yeast (3, 4). However, AtNHX1 complements a yeast mutant defective in ScNHX1 and restores cation compartmentalization (5).Improving the salt tolerance of crop plants is an important goal in biotechnology. In addition to the mechanisms by which a cell can cope with increased concentrations of toxic cations, it is important to know the identity of salt-sensitive cellular targets. Only a few key processes have been identified. In yeast, HAL2, an inositol phosphatase that catalyzes the dephosphorylation of 3′-phosphoadenosine-5′-phosphate to AMP, has been found to be inhibited by Li⁺ and Na⁺. Inhibition of HAL2 during salt stress results in the accumulation of 3′-phosphoadenosine-5′-phosphate in the cell, which has the potential to produce a variety of toxic effects, such as the inhibition of sulfotransferases and RNA-processing enzymes (6). Another possible target is the KEX2/furin family of proteases of the Golgi/secretory pathway. The activity of KEX2 in vitro has been shown to respond differently, depending on the alkali cation and concentration present in the medium (7). Here, we show that the endosomal system is an additional target for Na⁺ toxicity.The Golgi apparatus, trans-Golgi network, and endosome/prevacuolar compartment form a continuum where proteins and membranes are modified en route to their final destinations (8–10). The late endosome/prevacuolar compartment is considered a key point in intracellular vesicle and protein trafficking. In addition to being the previous stage for vacuolar sorted proteins and cargo, this is where both the exocytic and endocytic pathways converge (10, 11). Ion homeostasis in these organelles is increasingly regarded as an important feature for intracellular transport processes (12–15). In particular, K⁺ concentration may regulate the activity and specificity of enzymes modifying proteins posttranslationally, such as the above mentioned KEX2/furin protease family (7). Lumenal pH has been reported also to regulate selective protein aggregation in secretory vesicles (12). In this respect, it is noteworthy that yeast nhx1 mutants have been characterized as class E vps mutants with impaired vacuole biogenesis and protein sorting (15).AtNHX1 is thought to increase salt tolerance in plants through the intracellular compartmentation of Na⁺. However, using purified protein, it has been shown that this antiporter can exchange H⁺ for K⁺, Na⁺, or Li⁺, albeit the last one with lower affinity (16). The poor K⁺/Na⁺ selectivity raises the question of whether Na⁺ transport is the primary function of AtNHX1 in plant cells and if AtNHX1 is amenable to selection of better alleles for salt tolerance. Mutagenesis of cation transporters has proved to be a valuable tool to obtain alleles with modified transport activities (17, 18). At the same time, this provides information about the important amino acid residues that affect the mechanism of protein function. In this work, we sought to produce hypermorphic AtNHX1 alleles conferring greater salt tolerance, by either improved Na⁺/K⁺ discrimination or altered protein regulation. We show here that nhx1-deficient yeast cells that express mutated forms of AtNHX1 display improved halotolerance compared with cells that express the wild-type AtNHX1. The mutations responsible for these changes were scattered throughout the hydrophobic N terminus of the protein, and their effect was to introduce bulkier side chain amino acids. Surprisingly, the result of these mutations was not increased compartmentalization of toxic alkali cations. Instead, all of these mutants showed a decreased content of Li⁺ (a tracer for Na⁺), whereas full amounts of K⁺ were retained. Biochemical characterization of a selected mutant transporter showed greater cation discrimination favoring K⁺ transport. AtNHX1 is localized to the vacuole and late endosome/prevacuolar compartment. Together, these results suggest that the endomembrane system is a cellular target of Na⁺ intoxication.     

A Quantitative Study of the Hog1 MAPK Response to Fluctuating Osmotic Stress in Saccharomyces cerevisiae

Background

Yeast cells live in a highly fluctuating environment with respect to temperature, nutrients, and especially osmolarity. The Hog1 mitogen-activated protein kinase (MAPK) pathway is crucial for the adaption of yeast cells to external osmotic changes.

Methodology/Principal Findings

To better understand the osmo-adaption mechanism in the budding yeast Saccharomyces cerevisiae, we have developed a mathematical model and quantitatively investigated the Hog1 response to osmotic stress. The model agrees well with various experimental data for the Hog1 response to different types of osmotic changes. Kinetic analyses of the model indicate that budding yeast cells have evolved to protect themselves economically: while they show almost no response to fast pulse-like changes of osmolarity, they respond periodically and are well-adapted to osmotic changes with a certain frequency. To quantify the signal transduction efficiency of the osmo-adaption network, we introduced a measure of the signal response gain, which is defined as the ratio of output change integral to input (signal) change integral. Model simulations indicate that the Hog1 response gain shows bell-shaped response curves with respect to the duration of a single osmotic pulse and to the frequency of periodic square osmotic pulses, while for up-staircase (ramp) osmotic changes, the gain depends on the slope.

Conclusions/Significance

The model analyses suggest that budding yeast cells have selectively evolved to be optimized to some specific types of osmotic changes. In addition, our work implies that the signaling output can be dynamically controlled by fine-tuning the signal input profiles.