Purification, gene cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces cerevisiae.

Saccharomyces cerevisiae S-II was purified to near homogeneity as a protein stimulating RNA polymerase II. Four of seven lysyl endopeptidase-digested fragments of S-II were located in the PPR2 sequence reported previously. Analysis of a genomic clone of S-II revealed that S-II and PPR2 are the same protein consisting of 309 amino acid residues, and frame shifts were found in the sequence of PPR2 gene reported previously. Yeast S-II and mouse S-II showed high similarity in their amino acid sequences, especially in their amino-terminal and carboxyl-terminal regions. A gene disruption experiment showed that an S-II null mutant was not lethal under usual growth conditions, indicating that S-II is not essential for the growth of yeast.     

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.     

PY motifs of Rod1 are required for binding to Rsp5 and for drug resistance

In Saccharomyces cerevisiae, the overexpression of ROD1 confers resistance to o-dinitrobenzene (o-DNB), a representative of target drugs of glutathione S-transferase. The roles of Rod1 in drug resistance have remained to be determined. We isolated the rog3 mutation as a suppressor mutation of the temperature sensitivity of the strain, in that two of the total four glycogen synthase kinase 3 homologs were deleted. Rog3 is homologous to Rod1, and its overexpression also conferred resistance to o-DNB. Furthermore, these two proteins have PY-motifs, and bound to Rsp5, a hect-type ubiquitin ligase. The rsp5-101 mutant showed sensitivity to o-DNB as did the rod1 mutant, a mutant Rod1 containing altered PY motifs was defective in ability to bind to Rsp5 and in conferring o-DNB resistance. These results suggest that interaction of Rod1 and Rsp5 is important for drug resistance.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.     

Ric1p and the Ypt6p GTPase function in a common pathway required for localization of trans-Golgi network membrane proteins

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.     

Yeast glycogen synthase kinase 3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs. The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3beta and by an osmotic stabilizer. Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1. We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant. Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated. We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants. Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3. Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced. These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.     

A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins.

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

CBT1 interacts genetically with CBP1 and the mitochondrially encoded cytochrome b gene and is required to stabilize the mature cytochrome b mRNA of Saccharomyces cerevisiae

Mutation of a CCG sequence in the 5'-untranslated region of the mitochondrially encoded cytochrome b mRNA in Saccharomyces cerevisiae results in destabilization of the message and respiratory deficiency of the mutant strain. This phenotype mimics that of a mutation in the nuclear CBP1 gene. Here it is shown that overexpression of the nuclear CBT1 gene, due to a transposon insertion in the 5'-untranslated region, rescues the respiratory defects resulting from mutating the CCG sequence to ACG. Overexpressing alleles of CBT1 are allelic to soc1, a previously isolated suppressor of cbp1ts-induced temperature sensitivity of respiratory growth. Quantitative primer extension analysis indicated that cbt1 null strains have defects in 5'-end processing of precursor cytochrome b mRNA to the mature form. Cbt1p is also required for stabilizing the mature cytochrome b mRNA after 5' processing.     

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Delta strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Delta strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.     

Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation

To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or non-specific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.     

A Conditional Allele of the Saccharomyces Cerevisiae Hop1 Gene Is Suppressed by Overexpression of Two Other Meiosis-Specific Genes: Red1 and Rec104

The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase. Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele. Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628. The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids. Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis. The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene. Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact. The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.     

BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

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.     

A conditional U5 snRNA mutation affecting pre-mRNA splicing and nuclear pre-mRNA retention identifies SSD1/SRK1 as a general splicing mutant suppressor.

A combination of point mutations disrupting both stem 1 and stem 2 of U5 snRNA (U5AI) was found to confer a thermosensitive phenotype in vivo. In a strain expressing U5AI, pre-mRNA splicing was blocked before the first step through an inability of the mutant U5 snRNA to efficiently associate with the U4/U6 di-snRNP. Formation of early splicing complexes was not affected in extracts prepared from U5 snRNA mutant cells, while the capacity of these extracts to splice a pre-mRNA in vitro was greatly diminished. In addition, significant levels of a translation product derived from intron containing pre-mRNAs could be detected in vivo. The SSD1/SRK1 gene was identified as a multi-copy suppressor of the U5AI snRNA mutant. Single copy expression of SSD1/SRK1 was sufficient to suppress the thermosensitive phenotype, and high copy expression partially suppressed the splicing and U4/U6.U5 tri-snRNP assembly pheno-types. SSD1/SRK1 also suppressed thermosensitive mutations in the Prp18p and U1-70K proteins, while inhibiting growth of the cold sensitive U1-4U snRNA mutant at 30 degrees C. Thus we have identified SSD1/SRK1 as a general suppressor of splicing mutants.