Parallel regulation of membrane trafficking and dominant-negative effects by misrouted gonadotropin-releasing hormone receptor mutants

Gonadotropin-releasing hormone (GnRH) receptor mutants from patients with hypogonadotropic hypogonadism are frequently misrouted proteins that exert a dominant-negative (DN) effect on human (h) wild-type (WT) receptor, due to oligomerization and retention in the endoplasmic reticulum. Pharmacologic chaperones restore correct folding, rescuing mutants and WT receptor from this oligomer. Rat WT retains the ability to oligomerize (since human and mouse mutants exert a DN effect on rat (r) WT sequence) but, unlike human or mouse, escapes the DN effect of GnRH receptor (Gn-RHR) mutants because rGnRHR mutants route to the plasma membrane with higher efficiency than mouse or human mutants. These distinct behaviors of mouse and rat GnRHRs (distinguished by only four semi- or non-conservative amino acid differences) led us to assess the role of each amino acid. The difference in both routing and the DN effect appears mediated primarily by Ser(216) in the rGnRHR. The homologous amino acid in the hGn-RHR is also Ser and is compensated for by the primate-unique insertion of Lys(191) that, alone, dramatically decreases routing of the receptor. These studies establish the relation between the DN effect and altered receptor trafficking and explain why hGnRHR is more susceptible to defective trafficking by disease-related point mutations than rodent counterparts.     

Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta-arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.     

Rescue of calcium-sensing receptor mutants by allosteric modulators reveals a conformational checkpoint in receptor biogenesis

The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating G(q)- and G(i)-linked signaling in the parathyroid, kidney, and intestine. CaR is ubiquitinated by the E3 ligase dorfin and degraded via the endoplasmic reticulum-associated degradation pathway (Huang, Y., Niwa, J., Sobue, G., and Breitwieser, G. E. (2006) J. Biol. Chem. 281, 11610-11617). Here we provide evidence for a conformational or functional checkpoint in CaR biogenesis using two complementary approaches. First we characterized the sensitivity of loss- or gain-of-function CaR mutants to proteasome inhibition by MG132. The stabilization of loss-of-function mutants and insensitivity of gain-of-function mutants to MG132 suggests that receptor sensitivity to calcium influences susceptibility to proteasomal degradation. Second, we used the allosteric activator NPS R-568 and antagonist NPS 2143 to promote the active and inactive conformations of wild type CaR, respectively. Overnight culture in NPS R-568 increased expression of CaR, whereas NPS 2143 had the opposite effect. NPS R-568 and NPS 2143 differentially regulated maturation and cell surface expression of wild type CaR, directly affecting maximal signaling responses. NPS R-568 rescued expression of loss-of-function CaR mutants, increasing plasma membrane expression and ERK1/2 phosphorylation in response to 5 mM Ca(2+). Disorders of calcium homeostasis caused by CaR mutations may therefore result from altered receptor biogenesis independent of receptor function, i.e. a protein folding disorder. The allosteric modulators NPS R-568 and NPS 2143 not only alter CaR sensitivity to calcium and hence signaling but also modulate receptor expression.     

Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission

Summary This communication demonstrates the usefulness of the plamid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk^- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome.These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.     

The structure of Sky1p reveals a novel mechanism for constitutive activity

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.     

Isolation of SOS constitutive mutants of Escherichia coli

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.

     

Selection of constitutive tryptophanase hyperproducing mutants of Escherichia coli

Biotechnology Letters - Constitutive tryptophanase hyperproducing mutants of Escherichia coli were isolated. The specific enzyme activities of these mutants are 3–5 times higher than those of...     

An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae

Summary A sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast. The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae. Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.     

Selection of mandelamidase constitutive mutants in continuous culture ofPseudomonas putida

Summary A method is described for the isolation of mandelamidase constitutive mutants ofPseudomonas putida in continuous culture. Growth was nitrogen-limited and the nitrogen was provided either as inducing substrate (mandelamide) or non-inducing substrate (isobutyramide) alternately.     

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.     

Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants

Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.     

Dextransucrase constitutive mutants of Leuconostoc mesenteroides B-1299

Leuconostoc mesenteroides B-1299 dextrans are separated into two kinds: fraction L, which is precipitated by an ethanol concentration of 38%, and fraction S, which is precipitated at an ethanol concentration of 40%. Fraction S dextran contained 35% of -1,2 branch linkages, and fraction L contained 27% -1,2 branch linkage with 1% -1,3 branch linkages. We have isolated mutants constitutive for dextransucrase from L. mesenteroides NRRL B-1299 using ethyl methane sulfonate. The mutants produced extracellular as well as cell-associated dextransucrases on glucose media with higher activities (2.5–4.5 times) than what the parental strain produced on sucrose. Based on Penicillium endo-dextranase hydrolysis, mutant B-1299C dextransucrases produced slightly different dextrans when they were elaborated on a glucose medium and on a sucrose medium. Mutant B-1299CA dextransucrase elaborated on a glucose medium and on a sucrose medium synthesized the same dextran, although the dextran was different from those of other mutants and the parental strain. Mutant B-1299CB dextransucrase, elaborated on a glucose medium and on a sucrose medium, formed different dextrans. Differences in water solubility, susceptibility to endo-dextranase hydrolysis, and the physical appearance of the ethanol precipitated dextrans elaborated by different mutants grown on glucose media and sucrose media were found. All mutant dextransucrases elaborated on a glucose medium bound to Sephadex G-200. After activity staining of nondenaturing sodium dodecyl sulfate—polyacrylamide gel electrophoresis activity bands, 184 and 240 Kd for each enzyme preparation, although each dextransucrase formed different dextran(s).     

Fine characterization of OsPHO2 knockout mutants reveals its key role in Pi utilization in rice

Previous research using forward genetics approaches demonstrated that OsPHO2 regulates multiple phosphate-starvation responses in rice. In this work, we finely characterized two independent OsPHO2 knockout rice mutants under inorganic phosphate (Pi)-sufficient conditions. The ospho2 mutants exhibited defects in growth and reproductive development in the whole growing period. The cells in the elongation zone of ospho2 seedling roots were much shorter than those of the wild type. The phosphorus concentration in the blades of ospho2 mutants was 5.8-fold higher than those of wild-type plants, whereas it was only slightly higher in the sheaths, culms, spikelets, and seeds. Furthermore, Pi levels in the ospho2 mutants were highest in the oldest leaf and lowest in the youngest leaf, whereas there was no significant difference in the corresponding leaves of wild-type plants. These results suggest that ospho2 mutant phenotype results from a partial defect in Pi translocation and remobilization in the shoot of rice. This study thus provides evidence that OsPHO2, which functions at the downstream of OsPHF1, modulates Pi utilization by regulating the expression of Pht1 transporters in rice.     

In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.     

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryp-tophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3′-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.     

Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis.

Constitutive mutants for L-arabinose utilization were isolated from Bacillus subtilis 168T+ and showed resistance to D-fucose, a nonmetabolizable analog of L-arabinose. The mutations that conferred the constitutive phenotype (Arac) were mapped between cysB and hisA. All the mutants showed an isomerase activity which was reduced to 50 to 70% in the presence of L-arabinose and to 10% in the presence of glucose.