A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions.

To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.     

A conserved amino acid motif (R-X-G-R-R) in the Glut1 glucose transporter is an important determinant of membrane topology.

The Glut1 glucose transporter is one of over 300 members of the major facilitator superfamily of membrane transporters. These proteins are extremely diverse in substrate specificity and differ in their transport mechanisms. The two most common features shared by many members of this superfamily are the presence of 12 predicted transmembrane segments and an amino acid motif, R-X-G-R-R, present at equivalent positions within the cytoplasmic loops joining transmembrane segments 2-3 and 8-9. The structural and functional roles of the arginine residues within these motifs in Glut1 were investigated by expression of site-directed mutant transporters in Xenopus oocytes followed by analyses of intrinsic transport activity and the membrane topology of mutant glycosylation-scanning reporter Glut1 molecules. Substitution of lysine residues for the cluster of 3 arginine residues in each of the 2 cytoplasmic pentameric motifs of Glut1 revealed no absolute requirement for arginine side chains at any of the 6 positions for transport of 2-deoxyglucose. However, removal of the 3 positive charges at either site by substitution of glycines for the arginines completely abolished transport activity as the result of a local perturbation in the membrane topology in which the cytoplasmic loop was aberrantly translocated into the exoplasm along with the two flanking transmembrane segments. Substitution of lysines for the arginines had no affect on membrane topology. We conclude that the positive charges in the R-X-G-R-R motif form critical local cytoplasmic anchor points involved in determining the membrane topology of Glut1. These data provide a simple explanation for the presence of this conserved amino acid motif in hundreds of functionally diverse membrane transporters that share a common predicted membrane topology.     

The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli

To investigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro, we constructed a set of variants of the N-terminal domain of lambda repressor with a wide range of melting temperatures. Pulse-chase experiments showed that, within this set, the proteins that are most thermally stable have the longest intracellular half-lives and vice versa. Moreover, second-site mutations which act directly or indirectly to increase the thermodynamic stability of the native N-terminal domain were found to suppress the intracellular degradation of one of the unstable mutants. These data suggest that thermal stability is, indeed, a key determinant of the proteolytic susceptibility of this protein in the cell. It is not the sole determinant, however, as sequences at the extreme C terminus of the N-terminal domain can influence proteolytic sensitivity without affecting the stability of the native structure. We propose that the thermal stability of the N-terminal domain of lambda repressor is an important determinant of its proteolytic sensitivity because degradation proceeds primarily from the unfolded form and that sequence determinants within the unfolded chain influence whether the unfolded protein will be a good substrate for proteolytic enzymes.     

The translation initiation factor eIF1A is an important determinant in the tolerance to NaCl stress in yeast and plants

Protein synthesis is very sensitive to NaCl. However, the molecular targets responsible for this sensitivity have not been described. A cDNA library of the halotolerant plant sugar beet was functionally screened in a sodium-sensitive yeast strain. We obtained a cDNA clone (BveIF1A) encoding the eukaryotic translation initiation factor eIF1A. BveIF1A was able to partially complement the yeast eIF1A-deficient strain. Overexpression of the sugar beet eIF1A specifically increased the sodium and lithium salt tolerance of yeast. This phenotype was not accompanied by changes in sodium or potassium homeostasis. Under salt stress conditions, yeast cells expressing BveIF1A presented a higher rate of amino acid incorporation into proteins than control cells. In an in vitro protein synthesis system from wheat germ, the BveIF1A recombinant protein improved translation in the presence of NaCl. Finally, transgenic Arabidopsis plants expressing BveIF1A exhibited increased tolerance to NaCl. These results suggest that the translation initiation factor eIF1A is an important determinant of sodium tolerance in yeast and plants.     

Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme)

The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.     

The carboxyl-terminal region of human interferon gamma is important for biological activity: mutagenic and NMR analysis.

Deletion of nine amino acids from the carboxyl terminus of human IFN gamma (residues 138--146; LFRGRRASQ) resulted in a 7-fold increase in specific antiviral activity. Similar increases in receptor binding affinity were seen. Deletion of residues 136 and 137 (QM) had little additional effect, but removal of Ser135 resulted in a sharp drop in antiviral activity. Further removal of residues 133 and 134 (KR) lowered antiviral activity to 1% of the peak value. Comparison of the proton NMR spectra of selected deletions down to residue 132 showed that there was no significant change in the core protein structure. Deletions down to residue 125 had the same antiviral activity as those to 132, but changes could now be seen in the aromatic proton NMR spectrum of this shorter derivative. Substitution of the homologous murine sequence between residues 124 and 130 (human SPAAKTG; murine LPESSLR) resulted in only a small decrease in antiviral activity, further suggesting that the precise sequence in this region was not critical for activity. Ser135 was substituted with a number of other amino acids with little or no change in activity. The importance of the residues between 131 and 134 for biological activity was corroborated by mutagenesis, although some substitutions in this region were tolerated.     

Pan1p, an actin cytoskeleton-associated protein, is required for growth of yeast on oleate medium

Pan1p is a yeast actin cytoskeleton-associated protein localized in actin patches. It activates the Arp2/3 complex, which is necessary for actin polymerization and endocytosis. We isolated the pan1-11 yeast mutant unable to grow on oleate as a sole carbon source and, therefore, exhibiting the Oleate- phenotype. In addition, mutant cells are temperature-sensitive and grow more slowly on glycerol or succinate-containing medium but similarly to the wild type on ethanol, pyruvate or acetate-containing media; this indicates proper functioning of the mitochondrial respiratory chain. However, growth on ethanol medium is compromised when oleic acid is present. Cells show growth arrest in the apical growth phase, and accumulation of cells with abnormally elongated buds is observed. The growth defects of pan1-11 are suppressed by overexpression of the END3 gene encoding a protein that binds Pan1p. The morphology of peroxisomes and induction of peroxisomal enzymes are normal in pan1-11, indicating that the defect in growth on oleate medium does not result from impairment in peroxisome function. The pan1-11 allele has a deletion of a fragment encoding amino acids 1109-1126 that are part of (QPTQPV)7 repeats. Surprisingly, the independently isolated pan1-9 mutant, which expresses a truncated form of Pan1p comprising aa 1-859, is able to grow on all media tested. Our results indicate that Pan1p, and possibly other components of the actin cytoskeleton, are necessary to properly regulate growth of dividing cells in response to the presence of some alternative carbon sources in the medium.     

Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant.

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.     

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.     

Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.     

Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria-specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs.     

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.     

Lysine 270 in the third intracellular domain of the oxytocin receptor is an important determinant for G alpha(q) coupling specificity

To identify structural elements important to specific G alpha(q) coupling in the oxytocin receptor (OTR), intracellular domains were exchanged between OTR and G alpha(s)-coupled vasopressin V(2) receptors (V(2)Rs). Substitution of sequence from the second (2i) and third (3i) intracellular domains of V(2)R into comparable positions in OTR markedly reduced ligand affinity and resulted in a loss of G alpha(q) coupling. Substitution of the 2i domain of OTR into V(2)R decreased ligand affinity and vasopressin-stimulated adenylyl cyclase activity and only slightly increased phosphatidylinositide turnover. In contrast, substitution of the OTR3i domain into V(2)R produced a receptor chimera with high ligand affinity, decreased vasopressin-stimulated adenylyl cyclase activity, and markedly enhanced ligand-stimulated phosphatidylinositide turnover. The C-terminal 36 amino acids, but not the N-terminal 13 amino acids, of the OTR3i domain contained the determinants critical for enhanced activation of PLC. Mutation of a single lysine in the C-terminal OTR3i sequence to the corresponding V(2)R residue (valine) eliminated the enhanced ability of the V(2)R chimera to stimulate PLC but did not affect maximal adenylyl cyclase stimulation. Furthermore, mutation of this residue (K270) in wild-type OTR completely abolished the ability of the receptor to stimulate phosphatidylinositide turnover, with only a small reduction in ligand affinity. These data demonstrate that OTR K270 is critically important in the stimulation by OTR of phosphatidylinositide turnover and that this determinant can also increase this activity in the V(2)R chimera. Mutation of K270 also adversely affects the ability of OTR to stimulate ERK1/2 phosphorylation. Therefore, this residue plays an important role in the specificity of OTR/G alpha(q)/PLC coupling.     

Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.     

Stage of development is an important determinant in the effect of nitrate on photoassimilate (13C) partitioning in chicory (Cichorium intybus)

The effect of nitrogen supply to chicory plants on carbon partitioningbetween shoot, root and tuberized root was studied at differentstages of vegetative growth, using long-term ¹³CO₂ labelling-chaseexperiments. This approach was complemented by measurement ofstorage carbohydrates and activities of enzymes involved inroot sucrose metabolism (sucrose-sucrose fructosyl transferase(SST), sucrose synthase, invertase). In both young and matureplants, low resulted in a 30–35% decrease in ¹³C assimilation. However, the partitioningof ¹³C between shoot and root was affected differently at differentstages of development. In young plants, in which carbohydrateswere being used for structural root and shoot growth, neither¹³C shoot/¹³C root ratio nor root activities of the above enzymeswere modified by supply. In contrast, in mature plants storing large amounts of carbohydratesas fructan in the tuberized root, low caused the ratio to decrease from 0.6 to 0.2, despiteunchanged net flux of ¹³C from shoot to root. The extractableactivity of SST was elevated in mature plants, compared to youngplants, at both low and high , consistent with its role in fructan synthesis. However, matureplants grown at low exhibited SST activity double that of plants grown at high . From these results, it is concluded that the observeddecrease in shoot/root dry weight ratio at low supply is caused by increased utilization of carbohydratesfor storage due to elevated root SST activity. Key words: Chicory, nitrate, ¹³C, shoot/root ratio, fructans, SST     

Acylation of Galpha(13) is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation

Palmitoylation of alpha-subunits in heterotrimeric G proteins has become a research object of growing attention. Following our recent report on the acylation of the mono-palmitoylated Galpha(12) [Ponimaskin et al., FEBS Lett. 429 (1998) 370-374], we report here on the identification of three palmitoylation sites in the second member of the G(12) family, Galpha(13), and on the biological significance of fatty acids on the particular sites. Using mutants of alpha(13) in which the potentially palmitoylated cysteine residues (Cys) were replaced by serine residues, we find that Cys-14, Cys-18 and Cys-37 all serve as palmitoylation sites, and that the mutants lacking fatty acids are functionally defective. The following biological functions of Galpha(13) were found to be inhibited: coupling to the PAR1 thrombin receptor, cell transformation and actin stress fiber formation. Results from established assays for the above functions with a series of mutants, including derivatives of the constitutively active mutant Galpha(13)Q226L, revealed a graded inhibitory response on the above mentioned parameters. As a rule, it appears that palmitoylation of the N-proximal sites (e.g. Cys-14 and Cys-18) contributes more effectively to biological function than of the acylation site located more internally (Cys-37). However, the mutant with Cys-37 replaced by serine is more severely inhibited in stress fiber formation (80%) than in cell transformation (50%), pointing to the possibility of a differential involvement of the three palmitoylation sites in Galpha(13).