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T N Davis

Publications and source records attributed to T N Davis.

At least 37 records · Page 2Linked to original sources
PubMed1993

Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae.

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.

Amides↗
PubMed1993

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

Binding Sites↗
PubMed1993

A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins.

The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae. We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1. Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1. Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function. HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1. HCM1 is located on chromosome III, which was recently sequenced. Our results correct errors in the published DNA sequence. The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622. Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa. Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins. When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription. However, overexpression of HCM1 does not affect the expression of calmodulin. Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay. Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism.

Amino Acid Sequence↗
PubMed1992

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.

Calcium↗
PubMed1992

A temperature-sensitive calmodulin mutant loses viability during mitosis.

Although rare, a recessive temperature-sensitive calmodulin mutant has been isolated in Saccharomyces cerevisiae. The mutant carries two mutations in CMD1, isoleucine 100 is changed to asparagine and glutamic acid 104 is changed to valine. Neither mutation alone conferred temperature sensitivity. A single mutation that allowed production of an intact but defective protein was not identified. At the nonpermissive temperature, the temperature-sensitive mutant displayed multiple defects. Bud formation and growth was delayed, but this defect was not responsible for the temperature-sensitive lethality. Cells synchronized in G1 progressed through the cell cycle and retained viability until the movement of the nucleus to the neck between the mother cell and the large bud. After nuclear movement, less than 5% of the cells survived the first mitosis and could form colonies when returned to permissive conditions. The duplicated DNA was dispersed along the spindle, extending from mother to daughter cell. Cells synchronized in G2/M lost viability immediately upon the shift to the nonpermissive temperature. At a semipermissive temperature, the mutant showed approximately a 10-fold increase in the rate of chromosome loss compared to a wild-type strain. The mitotic phenotype is very similar to yeast mutants that are defective in chromosome disjunction. The mutant also showed defects in cytokinesis.

Calmodulin↗
PubMed1992

Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae.

Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.

Actins↗
PubMed1991

Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.

Amino Acid Sequence↗
PubMed1991

Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells.

Deletion of Glu-84, Glu-83 and Glu-84, or Ser-Glu-Glu-Glu (residues 81-84) from the central helix of mammalian calmodulin is known to result in a 5-7 times decrease in its apparent in vitro affinities for three calmodulin-dependent enzymes. However, based on in vitro experiments, it is difficult to estimate how these deletions might affect in vivo cellular function. The yeast Saccharomyces cerevisiae, which requires calmodulin for growth, provides an excellent system to evaluate these deletion proteins in vivo. Based on its ability to restore normal growth characteristics to yeast cells, mammalian calmodulin is functionally identical to the yeast protein; herein we evaluate the effect of deleting residues 84, 83 and 84, or 81-84 from the central helix. Sequences encoding the deletion proteins and an unaltered control sequence were introduced by means of a yeast shuttle vector and were expressed under control of the yeast calmodulin promoter. The deletion and control calmodulins are produced at levels similar to that observed for the yeast protein, and they completely restore normal growth characteristics. This result suggests that the regions deleted from the central helix are not critical for activation of any yeast calmodulin target normally required for cell growth or division. It is likely that there are twisting and shortening motions associated with the deletions from the central helix that alter significantly the spatial relationship between the two lobes of calmodulin. The abilities of the deletion calmodulins to restore completely normal growth characteristics to yeast cells suggest that the lobes of all the deletion proteins can still be appropriately positioned in calmodulin-target complexes. This is consistent with the hypothesis that the central helix of calmodulin is analogous to a flexible tether rather than to a rigid connector between the two lobes of the molecule.

Amino Acid Sequence↗
PubMed1989

Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable.

Yeast strains relying solely on vertebrate (Xenopus laevis) calmodulin, expressed under control of a yeast (GAL1) promoter, grew at the same rate as yeast cells containing their endogenous calmodulin. Therefore, the ability to perform essential functions has been conserved between yeast and vertebrate calmodulins, suggesting that calmodulin performs the same (or overlapping) roles in yeast as it does in higher eukaryotes. Successful substitution of vertebrate for yeast calmodulin also suggests that the two proteins can adopt similar conformations in vivo, despite the large number of amino acid differences between them (60 out of 148 residues). Strains overproducing either vertebrate or yeast calmodulin about 100-fold and a strain producing a normal level of yeast calmodulin were essentially indistinguishable in many characteristics, including microtubule distribution, rate of secretion, response to mating pheromone, sporulation, and adaptation to nutrient limitation. Calmodulin overproduction did not confer elevated resistance to a phenothiazine drug, trifluoperazine, thought to be a calmodulin-specific inhibitor. These results have important implications for understanding the role of calmodulin in intracellular calcium signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Isolation of the yeast calmodulin gene: calmodulin is an essential protein.

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.

Amino Acid Sequence↗
PubMed1986

Magnetic resonance imaging and ultrasonography in the antenatal evaluation of conjoined twins.

Magnetic resonance imaging and sonography were used as diagnostic aids in the antepartum evaluation of two sets of conjoined twins. Magnetic resonance imaging was performed at 20 weeks' estimated gestational age on thoracopagus/omphalopagus twins and on omphalopagus twins at 20 and 30 weeks. Magnetic resonance imaging is a complementary adjunct to ultrasonography and provides additional anatomically precise clinical data. The advantages of this noninvasive technique include a large diagnostic window allowing total fetal imaging with excellent resolution of tissue composition. Unlike computerized tomographic imaging, there is no associated radiation exposure. The principal disadvantages of magnetic resonance imaging at this time are the cost of the equipment and the lack of real-time imaging capability.

Adult↗
PubMed1985

An alkyl imidate labeling study of the organization of phospholipids and proteins in the lipid-containing bacteriophage PR4.

The structure of the lipid-containing bacteriophage PR4 was studied using two alkyl imidates, ethyl acetimidate (EAI), a reagent permeant to lipid bilayers and isethionyl acetimidate (IAI), which is impermeant to membranes. The virion is an icosahedral particle consisting of a protein coat surrounding a membrane of phospholipid and protein which in turn encloses the DNA genome. Upon exposure to the permeant reagent, EAI, 50% of the phosphatidylethanolamine (PE) molecules reacted rapidly (half-life less than 10 min). A similar fraction of the PE also reacted with IAI, the impermeant reagent. The remaining half of the PE molecules reacted slowly with EAI (half-life of 80 min) and failed to react with IAI. All of the phage proteins reacted with both EAI and IAI (except a DNA-associated protein which reacted only with EAI). These labeling results indicate that the phage membrane consists of a lipid bilayer and that at least a portion of each phage protein (except the DNA-associated protein) is exposed on the external face of the lipid bilayer. Several of the membrane proteins could be cross-linked either to the phage membrane PE after EAI treatment or to phage phosphatidylglycerol after periodate treatment. The major structural protein of the phage was readily cross-linked to PG but failed to cross-link to PE suggesting that the protein specifically interacts with PG.

Cross-Linking Reagents↗
PubMed1983

Nonsense mutants of the lipid-containing bacteriophage PR4.

Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated. One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles. We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells. Finally each complementation group was tested for the ability to lyse an Su- host. We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme.

Bacteriophages↗