A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus

SAR1, a gene that has been isolated as a multicopy suppressor of the yeast ER-Golgi transport mutant sec12, encodes a novel GTP-binding protein. Its nucleotide sequence predicts a 21-kD polypeptide that contains amino acid sequences highly homologous to GTP-binding domains of many ras-related proteins. Gene disruption experiments show that SAR1 is essential for cell growth. To test its function further, SAR1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SAR1 copy disrupted. This mutant grows normally in galactose medium but arrests growth 12-15 h after transfer to glucose medium. At the same time, mutant cells accumulate ER precursor forms of a secretory pheromone, alpha-mating factor, and a vacuolar enzyme, carboxypeptidase Y. We propose that Sec12p and Sarlp collaborate in directing ER-Golgi protein transport.     

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

Characterization of multicopy suppressor genes that complement a defect in the Wis1-Sty1 MAP kinase cascade involved in stress responses in Schizosaccharomyces pombe

The Wis1-Sty1 mitogen-activated protein (MAP) kinase cascade is one of the major signaling systems involved in a wide range of stress responses in Schizosaccharomyces pombe. It is known that Deltawis1 and Deltasty1 mutants exhibit highly pleiotropic phenotypes, including a phenotype of temperature sensitivity for growth. In this study, we screened multicopy suppressor genes that allow both the Deltawis1 and Deltasty1 mutants to grow simultaneously at a non-permissive temperature, 37 degrees C. Two such multicopy suppressors were cloned and characterized as sds23(+) and hxk2(+) genes. The former is known to specify a protein that functions as a multicopy suppressor for mutations of the PP1 protein phosphatase and the 20S cyclosome/anaphase-promoting complex (APC), and the latter encodes hexokinase 2. It was revealed that the multicopy sds231 gene restored a defect in the mating efficiency caused by the Deltawis1 and Deltasty1 mutations, whereas the multicopy hxk2(+) gene suppressed a phenotype of heat-shock sensitivity for growth of these mutant cells. These findings are discussed with special reference to the Wis1-Sty1 MAP kinase signaling pathway in S. pombe.     

The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.     

BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex.

A subset of the genes required for transport from the endoplasmic reticulum (ER) to the Golgi complex in Saccharomyces cerevisiae was found to interact genetically. While screening a yeast genomic library for genes complementing the ER-accumulating mutant bet1 (A. Newman and S. Ferro-Novick, J. Cell Biol. 105: 1587-1594, 1987), we isolated BET1 and BOS1 (bet one suppressor). BOS1 suppresses bet1-1 in a gene dosage-dependent manner, providing greater suppression when it is introduced on a multicopy vector than when one additional copy is present. The BET1 and BOS1 genes are not functionally equivalent; overproduction of BOS1 does not alleviate the lethality associated with disruption of BET1. We also identified a pattern of genetic interactions among these genes and another gene implicated in transport from the ER to the Golgi complex: SEC22. Overproduction of either BET1 or BOS1 suppresses the growth and secretory defects of the sec22-3 mutant over a wide range of temperatures. Further evidence for genetic interaction was provided by the finding that a bet1 sec22 double mutant is inviable. Another mutant which is blocked in transport from the ER to the Golgi complex, sec21-1, demonstrates a more limited ability to be suppressed by the BET1 gene. The interactions we observed are specific for genes required for transport from the ER to the Golgi complex. The products of the genes involved are likely to have a direct role in transport, as bet1-1 and sec22-3 begin to display their mutant phenotypes within 5 min of a shift to the restrictive temperature.     

Multicopy suppressors of the cold-sensitive phenotype of the pcsA68 (dinD68) mutation in Escherichia coli.

The Escherichia coli strain cs2-68 is a cold-sensitive (c) mutant that forms a long filamentous cell at 20 degrees C with a large nucleoid mass in its central region. We have recently shown that the pcsA68 mutation causing the cs phenotype is a single-base substitution within the dinD gene, a DNA damage-inducible gene which maps at 82 min. Since null mutants of the pcsA (dinD) gene are viable, with no discernible defect in cell growth, the cs phenotype is attributed to a toxic effect by the mutant protein. In an attempt to identify a target(s) for the toxic pcsA68 mutant protein, we screened for chromosomal fragments on multicopy plasmids that could suppress the cs phenotype. Three different BamHI fragments were found to suppress cold sensitivity, and the lexA, dinG, and dinI genes were identified to be responsible for the suppression in each fragment. DinG shares multiple motifs with many DNA helicases. The complete sequence of dinI revealed that DinI is a small protein of 81 amino acids. It is similar in size and sequence to ImpC of the Salmonella typhimurium plasmid TP110 and to a protein (ORFfs) of the retronphage phi R67, both of which are also under the control of LexA.     

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.     

A tRNA(TRP) gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8

The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223). Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b. ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis. In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1. COQ6 is essential for yeast coenzyme Q biosynthesis. We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons. The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported.     

Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA.

We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene.     

Understanding the growth phenotype of the yeast gcr1 mutant in terms of global genomic expression patterns

The phenotype of an organism is the manifestation of its expressed genome. The gcr1 mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available. Using DNA microarrays, the genomic expression patterns of wild-type and gcr1 mutant yeast growing on various media, with and without glucose, were compared. A total of 53 open reading frames (ORFs) were identified as GCR1 dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate. The GCR1-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be GCR1 dependent. In wild-type cultures, GCR1-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total. Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts. In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type. By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background. Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources. Thus, the severe growth inhibition observed in gcr1 mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.     

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.     

Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25

We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid. Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family. The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product. The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA. The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene. Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein. Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.     

Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.     

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).     

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating “shuttle” vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2⁺ allele has been cloned.