Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae.

The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.     

The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae.

The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.     

AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae.

We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.     

CYP1 (HAP1) regulator of oxygen-dependent gene expression in yeast. I. Overall organization of the protein sequence displays several novel structural domains

In the yeast Saccharomyces cerevisiae the CYP1 gene that modulates the expression of iso1-(CYC1) and iso2-cytochrome c (CYP3) structural genes gives rise to two classes of mutated alleles; one class, represented by CYP1-18, has opposite effects on CYC1 and CYP3, it reduces the expression of CYC1 while it stimulates that of CYP3. The other class, represented by cyp1-23 or the related allele hap1-1, reduces the expression of both CYC1 and CYP3 genes. Genetic data suggested that the CYP1 product is a positive regulator of the cytochrome c genes. The CYP1-18 allele has been cloned. We show here that the iso2 overproducer function of CYP1-18 is included in a 5300 base XhoI-PstI fragment. The sequence of this fragment reveals a unique, long, uninterrupted open reading frame of 4449 nucleotides able to encode a protein of 1483 amino acid residues. The predicted product of this open reading frame contains several interesting features. The N-terminal part of the protein resembles a nucleic acid-binding domain, in which two domains can be distinguished. The first is similar to a "finger" DNA binding motif, as found in TFIIIA and other regulatory proteins. The second consists of seven tandemly repeated sequences with a KCPVDH motif. Because of its structure, it is tempting to speculate that this region may act as a "redox sensor" folded around a metal atom or heme and involved in recognition of respiratory effectors. These two domains are separated by an "opa" sequence of 13 Gln residues. Implication of these domains for the function of CYP1-18 is discussed.     

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.     

Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac).

The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified. These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10). pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pac cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site. PacB extracts also contain a second P1 protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pac cleavage is yet to be determined.     

Comparison of S-layer secretion genes in freshwater caulobacters

Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus. All elaborate a crystalline protein surface (S) layer made up of protein monomers 100-193 kDa in size. We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100-108 kDa), medium (122-151 kDa), and large (181-193 kDa). All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C. crescentus, indicating that the S-layer proteins were all secreted by a type I system. The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals. Cross-expression studies showed that the 6 strains recognized secretion signals from C. crescentus and Pseudomonas aeruginosa and similarly that C. crescentus was able to secrete the S-layer protein C-terminus of 1 strain examined. Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion. Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size.     

Activation of caspase-like activity and poly (ADP-ribose) polymerase degradation during sporulation in Aspergillus nidulans

Mycelium vacuolization, protein degradation, and as the final stage autolysis, often accompanies developmental changes in fungi and similarities between autolysis and apoptosis have previously been suggested. Caspases are the key executors of apoptosis and in this study caspase-like activities were detected in protein extracts from Aspergillus nidulans during sporulation. This was shown by hydrolysis of the fluorescent DEVD- and IETD-AFC peptide substrates specific for caspase 3- and 8-like activities, respectively. These activities were repressed by the caspase 3 and 8 specific irreversible peptide inhibitors DEVD-fmk and IETD-fmk, but were not affected by the unspecific inhibitor E-64. Isoelectric focusing of protein extracts followed by activity staining revealed the presence of two bands with caspase-like activity. One of the proteins degraded both caspase 3 and caspase 8 specific substrates whereas the other only degraded the caspase 8 substrate. Searches in an A. nidulans genome database revealed two genes encoding metacaspase proteins with predicted sizes of 45 kDa that could be responsible for the measured caspase-like activities. The searches also found a single gene encoding a poly (ADP-ribose) polymerase (PARP) protein with a predicted size of 81 kDa. PARP is one of the known target proteins inactivated by caspase degradation in animal cells. Western blotting of fungal extracts using a bovine PARP antibody confirmed the presence of a fungal PARP-like protein of about 81 kDa. By Western blotting it was shown that this PARP-like protein band was present only at early time points until the start of conidia formation and the accompanying increase in caspase-like activity. Thereafter, a degradation product of about 60 kDa appeared indicating that the degradation of the fungal PARP-like protein was specific. The PARP antibody also recognized an 85 kDa protein band that was not degraded, and which conceivably represents a modified form of the 81 kDa PARP. Fungal extracts high in caspase-like activity could degrade both the fungal 81 kDa PARP and bovine PARP. In the presence of the caspase 3 inhibitor DEVD-fmk this degradation was delayed. Thus, as in animal apoptotic cells, caspase activities are involved in fungal mycelium self-activated proteolysis.     

The Cyc8 (Ssn6)-Tup1 corepressor complex is composed of one Cyc8 and four Tup1 subunits.

The Cyc8 (Ssn6)-Tup1 corepressor complex is required for repression in several important regulatory systems in yeast cells, including glucose repression and mating type. Cyc8-Tup1 is recruited to target genes by interaction with diverse repressor proteins that bind directly to DNA. Since the complex has a large apparent molecular mass of 1,200 kDa on nondenaturing gels (F. E. Williams, U. Varanasi, and R. J. Trumbly, Mol. Cell. Biol. 11:3307-3316, 1991), we used a variety of approaches to determine its actual subunit composition. Immunoprecipitation of epitope-tagged complex and reconstitution of the complex from in vitro-translated proteins demonstrated that only the Cyc8 and Tup1 proteins were present in the complex. Hydrodynamic properties showed that these proteins have unusually large Stokes radii, low sedimentation coefficients, and high frictional ratios, all characteristic of asymmetry which partly accounts for the apparent high molecular weight. Calculation of native molecular weights from these properties indicated that the Cyc8-Tup1 complex is composed of one Cyc8 subunit and four Tup1 subunits. This composition was confirmed by reconstitution of the complex from Cyc8 and Tup1 expressed in vitro and analysis by one- and two-dimensional gel electrophoresis.     

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.