Yeast calmodulin: structural and functional elements essential for the cell cycle.

The budding yeast Saccharomyces cerevisiae is a suitable organism for studying calmodulin function in cell proliferation. Genetic studies in yeast demonstrate that vertebrate calmodulin can functionally replace yeast calmodulin. In addition, expression of half of the yeast calmodulin molecule is found to be sufficient for cell growth. Characterization of conditional-lethal mutants of yeast calmodulin as well as the intracellular distribution of calmodulin have suggested that at least two cell cycle steps require calmodulin function. One is nuclear division and the other is the maintenance of cell polarity. A current focus is to understand which kinds of target proteins are involved in mediating the essential functions of yeast calmodulin in these processes. Thus far, three yeast enzymes whose activity is regulated by calmodulin have been identified.     

Functional expression of chicken calmodulin in yeast

The coding region of a chicken calmodulin cDNA was fused to a galactose-inducible GAL1 promoter, and an expression system was constructed in the yeast Saccharomyces cerevisiae. Expression of calmodulin was demonstrated by purifying the heterologously expressed protein and analyzing its biochemical properties. When the expression plasmid was introduced into a calmodulin gene (cmd1)-disrupted strain of yeast, the cells grew in galactose medium, showing that chicken calmodulin could complement the lesion of yeast calmodulin functionally. Repression of chicken calmodulin in the (cmd1)-disrupted strain caused cell cycle arrest with a G2/M nucleus, as observed previously with a conditional-lethal mutant of yeast calmodulin. These results suggest that the essential function of calmodulin for cell proliferation is conserved in cells ranging from yeast to vertebrate cells.     

A site-directed mutagenesis study of yeast calmodulin

A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.     

Protein A–calmodulin fusions: a novel approach for investigating calmodulin function in yeast

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.     

Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin

Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin. The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol). The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added. On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin. Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.     

The whole is not the simple sum of its parts in calmodulin from S. cerevisiae

Calmodulin is an essential Ca(2+)-binding protein involved in a multitude of cellular processes. The calmodulin sequence is highly conserved among all eukaryotic species; calmodulin from the yeast S. cerevisiae (yCaM) is the most divergent form, while still sharing 60% sequence identity with vertebrate calmodulin (vCaM). Although yCaM can be functionally substituted by vCaM in vivo, the two calmodulin proteins possess significantly different Ca(2+)-binding properties as well as abilities to activate vertebrate target enzymes in vitro. In addition, it has been observed that certain properties of the N-terminal and C-terminal domains of Ca(2+)-yCaM differ depending on whether they are in the context of the whole protein or isolated as half-molecule fragments. To investigate the structural basis for these differing properties, we have undertaken nuclear magnetic resonance (NMR) studies on yCaM and the two half-molecule fragments representing its two individual domains, yTr1(residues 1-76) and yTr2 (residues 75-146). We present direct evidence that the two domains of Ca(2+)-yCaM interact via their exposed hydrophobic surfaces. Thus, the Ca(2+)-bound form of yCaM exists in a novel compact structure in direct contrast to the well-established structure of Ca(2+)-vCaM comprised of two independent globular domains.     

Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.     

Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. An alpha-factor inducible gene.

A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes. The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.     

Quantitative genetic analysis in Saccharomyces cerevisiae using epistatic miniarray profiles (E-MAPs) and its application to chromatin functions

The use of the budding yeast Saccharomyces cerevisiae as a simple eukaryotic model system for the study of chromatin assembly and regulation has allowed rapid discovery of genes that influence this complex process. The functions of many of the proteins encoded by these genes have not yet been fully characterized. Here, we describe a high-throughput methodology that can be used to illuminate gene function and discuss its application to a set of genes involved in the creation, maintenance and remodeling of chromatin structure. Our technique, termed E-MAPs, involves the generation of quantitative genetic interaction maps that reveal the function and organization of cellular proteins and networks.     

Mathematical modeling of calcium homeostasis in yeast cells

In this study, based on currently available experimental observations on protein level, we constructed a mathematical model to describe calcium homeostasis in normally growing yeast cells (Saccharomyces cerevisiae). Simulation results show that tightly controlled low cytosolic calcium ion level can be a natural result under the general mechanism of gene expression feedback control. The calmodulin (a sensor protein) behavior in our model cell agrees well with relevant observations in real cells. Moreover, our model can qualitatively reproduce the experimentally observed response curve of real yeast cell responding to step-like disturbance in extracellular calcium ion concentration. Further investigations show that the feedback control mechanism in our model is as robust as it is in real cells.     

The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response.

The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle.     

Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression.     

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.     

Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.     

The ras oncogene--an important regulatory element in lower eucaryotic organisms.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.     

Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae

Pseudomonas putida benF, benK, benE1, and benE2 genes encode proteins belonging to benzoate transporter super family, but those functions have not yet been elucidated. In this study we analyzed the functions of these gene products using the yeast Saccharomyces cerevisiae. P. putida gene products expressed in yeast cells were localized to the yeast plasma membrane and were involved in taking up benzoate into the cells. According to the sensitivity of yeast cell-growth to benzoate, it is proposed that benK, benE1, and benE2 gene products function as transporters, that take up benzoate into the cells, whereas the benF gene product functions as an efflux pump of benzoate.     

A strange calmodulin of yeast

Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.     

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.