The yeast cell cycle.

This review focuses on the G1 regulation of the p34cdc2/CDC28 kinase by cyclin-like proteins, which has substantially altered our understanding of cell cycle control. We discuss advances in elucidating the molecular composition of the mitotic apparatus, an essential step in understanding its cell-cycle-dependent assembly and functions.

Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein.

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.

The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.

Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase.

The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: the 69-kilodalton (kD) catalytic subunit of the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) and a 50-kD protein. The 69-kD subunit is encoded by the 5' and 3' thirds of the TFP1 coding region, whereas the 50-kD protein is encoded by the central third. Evidence is presented that both the 69-kD and 50-kD proteins are obtained from a single translation product that is cleaved to release the 50-kD protein and spliced to form the 69-kD subunit.

The TPR snap helix: a novel protein repeat motif from mitosis to transcription.

The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi-domain proteins all contain tandemly arranged repeats of a 34-amino acid motif that are presumed to form helix-turn structures, each with a 'knob' and 'hole', acting as helix-associating domains.

The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.

The abundance of cell cycle regulatory protein Cdc4p is controlled by interactions between its F box and Skp1p.

Posttranslational modification of a protein by ubiquitin usually results in rapid degradation of the ubiquitinated protein by the proteasome. The transfer of ubiquitin to substrate is a multistep process. Cdc4p is a component of a ubiquitin ligase that tethers the ubiquitin-conjugating enzyme Cdc34p to its substrates. Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, we have sought evidence of mechanisms that modulate F-box protein regulation. Here we demonstrate that the abundance of Cdc4p is subject to control by a peptide segment that we term the R-motif (for "reduced abundance"). Furthermore, we show that binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities.

CDC33 encodes mRNA cap-binding protein eIF-4E of Saccharomyces cerevisiae.

The bcy1 mutation makes the cdc33 start mutant arrest at random points in the cell cycle instead of only at G1. We cloned and sequenced CDC33. This coding sequence is identical to that of the gene encoding the Saccharomyces cerevisiae 24-kilodalton mRNA cap-binding protein, eIF-4E.

Phosphorylation of nuclear MyoD is required for its rapid degradation.

MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).

Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.

The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability.

Ubiquitin-mediated degradation plays a crucial role in many fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin ligase complexes, called SCF complexes, found throughout eukaryotes, is involved in a variety of biological pathways. In Saccharomyces cerevisiae, an SCF complex contains a common set of components, namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a variable component called an F-box protein. The F- box is a approximately 40-amino-acid motif that allows the F-box protein to bind Skp1p. Each SCF complex recognizes different substrates according to which F-box protein is associated with the complex. In yeasts, three SCF complexes have been demonstrated to associate with the ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity. F-box proteins are not abundant and are unstable. As part of the SCF(Met30p) complex, the F-box protein Met30p represses methionine biosynthetic gene expression when availability of L-methionine is high. Here we demonstrate that in vivo SCF(Met30p) complex activity can be regulated by the abundance of Met30p. Furthermore, we provide evidence that Met30p abundance is regulated by the availability of L-methionine. We propose that the cellular responses mediated by an SCF complex are directly regulated by environmental conditions through the control of F-box protein stability.

Mouse receptor interacting protein 3 does not contain a caspase-recruiting or a death domain but induces apoptosis and activates NF-kappaB.

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.

Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells. We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen. These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase. Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis. Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen. Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase. These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell. The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase.

Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.

The identification of transposon-tagged mutations in essential genes that affect cell morphology in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphology-elongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis.

Genetic interactions between REG1/HEX2 and GLC7, the gene encoding the protein phosphatase type 1 catalytic subunit in Saccharomyces cerevisiae.

Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7Y-170 allele, lack of growth at 14 degrees. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation.