The ATPase and protease domains of yeast mitochondrial Lon: Roles in proteolysis and respiration-dependent growth

The ATP-dependent Lon protease of Saccharomyces cerevisiae mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth. Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex assembly. To understand the influence of Lon’s ATPase and protease activities on these functions, we examined several Lon mutants for their ability to complement defects of Lon-deleted yeast cells. We also developed a rapid procedure for purifying yeast Lon to homogeneity to study the enzyme’s activities and oligomeric state. A point mutation in either the ATPase or the protease site strongly inhibited the corresponding activity of the pure protein but did not alter the protein’s oligomerization; when expressed at normal low levels neither of these mutant enzymes supported respiration-dependent growth of Lon-deleted cells. When the ATPase- or the protease-containing regions of Lon were expressed as separate truncated proteins, neither could support respiration-dependent growth of Lon-deleted cells; however, coexpression of these two separated regions sustained wild-type growth. These results suggest that yeast Lon contains two catalytic domains that can interact with one another even as separate proteins, and that both are essential for the different functions of Lon.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.

Interaction of the E1A Oncoprotein with Yak1p, a Novel Regulator of Yeast Pseudohyphal Differentiation, and Related Mammalian Kinases

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiff-man syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4+ T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

Futile transmembrane NH4+ cycling: A cellular hypothesis to explain ammonium toxicity in plants

Most higher plants develop severe toxicity symptoms when grown on ammonium (NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}) as the sole nitrogen source. Recently, NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity have been extensively sought, the primary events conferring it at the cellular level are not understood. Using a high-precision positron tracing technique, we here present a cell-physiological characterization of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} acquisition in two major cereals, barley (Hordeum vulgare), known to be susceptible to toxicity, and rice (Oryza sativa), known for its exceptional tolerance to even high levels of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}. We show that, at high external NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} concentration ([NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o), barley root cells experience a breakdown in the regulation of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} influx, leading to the accumulation of excessive amounts of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} in the cytosol. Measurements of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}, reveal that, at elevated [NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o, barley cells engage a high-capacity NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}-efflux system that supports outward NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} fluxes against a sizable gradient. Ammonium efflux is shown to constitute as much as 80% of primary influx, resulting in a never-before-documented futile cycling of nitrogen across the plasma membrane of root cells. This futile cycling carries a high energetic cost (we record a 40% increase in root respiration) that is independent of N metabolism and is accompanied by a decline in growth. In rice, by contrast, a cellular defense strategy has evolved that is characterized by an energetically neutral, near-Nernstian, equilibration of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} at high [NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o. Thus our study has characterized the primary events in NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} nutrition at the cellular level that may constitute the fundamental cause of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity in plants.

Arabidopsis Roots and Shoots Have Different Mechanisms for Hypoxic Stress Tolerance

Arabidopsis has inducible responses for tolerance of O2 deficiency. Plants previously exposed to 5% O2 were more tolerant than the controls to hypoxic stress (0.1% O2 for 48 h) in both roots and shoots, but hypoxic acclimation did not improve tolerance to anoxia (0% O2). The acclimation of shoots was not dependent on the roots: increased shoot tolerance was observed when the roots of the plants were removed. An adh (alcohol dehydrogenase) null mutant did not show acclimation of the roots but retained the shoot survival response. Abscisic acid treatment also differentiated the root and shoot responses; pretreatment induced root survival in hypoxic stress conditions (0.1% O2) but did not induce any increase in the survival of shoots. Cycloheximide blocked both root and shoot acclimation, indicating that both acclimation mechanisms are dependent on protein synthesis.

Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions

Compelling evidence has accumulated over the last several years from our laboratory, as well as others, indicating that central hyperactive states resulting from neuronal plastic changes within the spinal cord play a critical role in hyperalgesia associated with nerve injury and inflammation. In our laboratory, chronic constriction injury of the common sciatic nerve, a rat model of neuropathic pain, has been shown to result in activation of central nervous system excitatory amino acid receptors and subsequent intracellular cascades including protein kinase C translocation and activation, nitric oxide production, and nitric oxide-activated poly(ADP ribose) synthetase activation. Similar cellular mechanisms also have been implicated in the development of tolerance to the analgesic effects of morphine. A recently observed phenomenon, the development of “dark neurons,” is associated with both chronic constriction injury and morphine tolerance. A site of action involved in both hyperalgesia and morphine tolerance is in the superficial laminae of the spinal cord dorsal horn. These observations suggest that hyperalgesia and morphine tolerance may be interrelated at the level of the superficial laminae of the dorsal horn by common neural substrates that interact at the level of excitatory amino acid receptor activation and subsequent intracellular events. The demonstration of interrelationships between neural mechanisms underlying hyperalgesia and morphine tolerance may lead to a better understanding of the neurobiology of these two phenomena in particular and pain in general. This knowledge may also provide a scientific basis for improved pain management with opiate analgesics.

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos1

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat1

The application of a moderate water deficit (water potential of −1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (−1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, β-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Accuracy of lesion bypass by yeast and human DNA polymerase η

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10−2 to 10−3 opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10−2 to 10−3 relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast.

Pseudohyphal differentiation in Saccharomyces cerevisiae was first described as a response of diploid cells to nitrogen limitation. Here we report that haploid and diploid starch-degrading S. cerevisiae strains were able to switch from a yeast form to a filamentous pseudohyphal form in response to carbon limitation in the presence of an ample supply of nitrogen. Two genes, MSS10 and MUC1, were cloned and shown to be involved in pseudohyphal differentiation and invasive growth. The deletion of MSS10 resulted in extremely reduced amounts of pseudohyphal differentiation and invasive growth, whereas the deletion of MUC1 abolished pseudohyphal differentiation and invasive growth completely. Mss10 appears to be a transcriptional activator that responds to nutrient limitation and coregulates the expression of MUC1 and the STA1-3 glucoamylase genes, which are involved in starch degradation. MUC1 encodes a 1367-amino acid protein, containing several serine/threonine-rich repeats. Muc1 is a putative integral membrane-bound protein, similar to mammalian mucin-like membrane proteins that have been implicated to play a role in the ability of cancer cells to invade other tissues.

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Functional expression of the Schizosaccharomyces pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae.

In the fission yeast, Schizosaccharomyces pombe, tolerance to high sodium and lithium concentrations requires the functioning of the sod2, Na+/H+ antiporter. We have directly measured the activity of this antiporter and demonstrated reconstitution of the activity in gene deletion strains. In addition, we have shown that it can be transferred to, and its antiporter activity detected in, the budding yeast, Saccharomyces cerevisiae, where it also confers sodium and lithium tolerance. Proton flux through the S. pombe Na+/H+ antiporter was directly measured using microphysiometry. The direction of transmembrane proton flux mediated by this antiporter was reversible, with protons being imported or exported in response to the external concentration of sodium. This bidirectional activity was also detected in S. cerevisiae strains expressing sod2 and expression of this gene complemented the sodium and lithium sensitivity resulting from inactivation of the ENA1/PMR2 encoded Na+-exporting ATPases. This suggests that antiporters or sodium pumps can be utilized interchangeably by S. cerevisiae to regulate internal sodium concentration. Potent inhibitors of mammalian Na+/H+ exchangers were found to have no effect on sod2 activity. The proton flux mediated by sod2 was also found to be unaffected by perturbation of membrane potential or the plasma membrane proton gradient.