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G C Johnston

Publications and source records attributed to G C Johnston.

At least 19 recordsLinked to original sources
PubMed2001

The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network.

Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

ADP-Ribosylation Factors↗
PubMed2001

A bipartite yeast SSRP1 analog comprised of Pob3 and Nhp6 proteins modulates transcription.

The FACT complex of vertebrate cells, comprising the Cdc68 (Spt16) and SSRP1 proteins, facilitates transcription elongation on a nucleosomal template and modulates the elongation-inhibitory effects of the DSIF complex in vitro. Genetic findings show that the related yeast (Saccharomyces cerevisiae) complex, termed CP, also mediates transcription. The CP components Cdc68 and Pob3 closely resemble the FACT components, except that the C-terminal high-mobility group (HMG) box domain of SSRP1 is not found in the yeast homolog Pob3. We show here that Nhp6a and Nhp6b, small HMG box proteins with overlapping functions in yeast, associate with the CP complex and mediate CP-related genetic effects on transcription. Absence of the Nhp6 proteins causes severe impairment in combination with mutations impairing the Swi-Snf chromatin-remodeling complex and the DSIF (Spt4 plus Spt5) elongation regulator, and sensitizes cells to 6-azauracil, characteristic of elongation effects. An artificial SSRP1-like protein, created by fusing the Pob3 and Nhp6a proteins, provides both Pob3 and Nhp6a functions for transcription, and competition experiments indicate that these functions are exerted in association with Cdc68. This particular Pob3-Nhp6a fusion protein was limited for certain Nhp6 activities, indicating that its Nhp6a function is compromised. These findings suggest that in yeast cells the Cdc68 partners may be both Pob3 and Nhp6, functioning as a bipartite analog of the vertebrate SSRP1 protein.

Carrier Proteins↗
PubMed2000

TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathway and plays a role in the developmental process of sporulation.

PTEN/MMAC1/TEP1 (PTEN, phosphatase deleted on chromosome ten; MMAC1, mutated in multiple advanced cancers; TEP1, tensin-like phosphatase) is a major human tumor suppressor gene whose suppressive activity operates on the phosphatidylinositol pathway. A single homologue of this gene, TEP1 (YNL128w), exists in the budding yeast Saccharomyces cerevisiae. Yeast strains deleted for TEP1 exhibit essentially no phenotype in haploids; however, diploids exhibit resistance to the phosphatidylinositol-3-phosphate kinase inhibitor wortmannin and to lithium ions. Although rates of cancer increase with age, neither tep1 haploids nor diploids have altered life spans. TEP1 RNA is present throughout the cell cycle, and levels are dramatically up-regulated during meiotic development. Although homozygous tep1 mutants initiate the meiotic program and form spores with wild-type kinetics, analysis of the spores produced in tep1 mutants indicates a specific defect in the trafficking or deposition of dityrosine, a major component of yeast spore walls, to the surface. Introduction of a common PTEN mutation found in human tumors into the analogous position in Tep1p produces a nonfunctional protein based on in vivo activity. These studies implicate Tep1p in a specific developmental trafficking or deposition event and suggest that Tep1p, like its mammalian counterpart, impinges on the phosphatidylinositol pathway.

Androstadienes↗
PubMed1999

Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function.

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER.

ADP-Ribosylation Factor 1↗
PubMed1998

Characterization of the CP complex, an abundant dimer of Cdc68 and Pob3 proteins that regulates yeast transcriptional activation and chromatin repression.

The Cdc68 protein of the yeast Saccharomyces cerevisiae is an essential nuclear protein that has been shown to be necessary for the trans-activation of many genes as well as for the maintenance of chromatin-mediated repression in the absence of trans-activation. These activities implicate the Cdc68 protein in the regulation of chromatin structure and/or function. Here we report that Cdc68 is found in association with another essential nuclear protein, Pob3, in what we term the CP complex. This dimer of Cdc68 with Pob3 is stable to partial purification, so that the functions of gene activation and repression that are assigned to Cdc68 are likely to be properties of the CP complex. The CP complex is highly abundant, suggesting that it may be widespread throughout chromatin.

Carrier Proteins↗
PubMed1998

A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation.

Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5'-CUG anticodon (tRNACUG). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.

Base Sequence↗
PubMed1998

The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Adenosine Triphosphatases↗
PubMed1996

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

ADP-Ribosylation Factor 1↗
PubMed1996

Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase.

The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.

Amino Acid Sequence↗
PubMed1995

Efficient translation of an SSA1-derived heat-shock mRNA in yeast cells limited for cap-binding protein and eIF-4F.

Eukaryotic mRNA molecules have a 5' cap structure that is recognized by the cap-binding component of translation initiation factor eIF-4F during protein synthesis. In the budding yeast Saccharomyces cerevisiae this cap-binding protein is encoded by the CDC33 gene. We report here that decreased global translation initiation in cdc33 mutant cells has virtually no effect on the translation of mRNA from the SSA1-lacZ chimeric gene, comprised of yeast SSA1 hsp70 gene transcription and translation initiation sequences fused in-frame to the bacterial lacZ gene. When global translation initiation was limited in cdc33 mutant cells, Ssa1-LacZ polypeptide synthesis was increased relative to total protein synthesis, and the beta-galactosidase activity of the Ssa1-LacZ fusion protein was induced to wild-type levels. The normal rate of Ssa1-LacZ polypeptide synthesis in mutant cells was maintained by normal levels of SSA1-lacZ mRNA. Furthermore, in cdc33 mutant cells, the size of polysomes containing SSA1-lacZ mRNA was unaffected, while polysomes containing other specific mRNAs were smaller. Efficient Ssa1-LacZ polypeptide synthesis was also seen during eIF-4F limitation produced by disruption of the TIF4631 gene, encoding the large eIF-4F subunit. All of these findings indicate efficient SSA1-lacZ mRNA usage under conditions of globally impaired translation initiation due to eIF-4F limitation.

Eukaryotic Initiation Factor-4F↗
PubMed1995

Mutations sensitizing yeast cells to the start inhibitor nalidixic acid.

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected, These aro7 alleles demonstrate 'recruitment', by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.

Cell Cycle↗
PubMed1995

Sug1 modulates yeast transcription activation by Cdc68.

The Cdc68 protein is required for the transcription of a variety of genes in the yeast Saccharomyces cerevisiae. In a search for proteins involved in the activity of the Cdc68 protein, we identified four suppressor genes in which mutations reverse the temperature sensitivity caused by the cdc68-1 allele. We report here the molecular characterization of mutations in one suppressor gene, the previously identified SUG1 gene. The Sug1 protein has been implicated in both transcriptional regulation and proteolysis. sug1 suppressor alleles reversed most aspects of the cdc68-1 mutant phenotype but did not suppress the lethality of a cdc68 null allele, indicating that sug1 suppression is by restoration of Cdc68 activity. Our evidence suggests that suppression by sug1 is unlikely to be due to increased stability of mutant Cdc68 protein, despite the observation that Sug1 affected proteolysis of mutant Cdc68. We report here that attenuated Sug1 activity strengthens mutant Cdc68 activity, whereas increased Sug1 activity further inhibits enfeebled Cdc68 activity, suggesting that Sug1 antagonizes the activator function of Cdc68 for transcription. Consistent with this hypothesis, we find that Sug1 represses transcription in vivo.

Adenosine Triphosphatases↗
PubMed1995

Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

Alleles↗
PubMed1994

A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation.

The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary-phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel 'Zn-finger' motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn-finger proteins.

Amino Acid Sequence↗
PubMed1994

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants.

The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42-46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristic of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna1 = cdc22, dna6 = cdc34, dna19 = cdc36, and dna39 = dbf3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhaustive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.

Alleles↗
PubMed1993

Yeast prt1 mutations alter heat-shock gene expression through transcript fragmentation.

The inhibition of translation initiation by modification or mutation of initiation factors can lead to disproportionate effects on gene expression. Here we report disproportionate decreases in gene expression in cells with mutated Prt1 activity. The PRT1 gene product of the budding yeast Saccharomyces cerevisiae is necessary for translation initiation and is thought to be a component of initiation factor 3. At a restrictive temperature the prt1-1 mutation, in addition to decreasing global protein synthesis, caused disproportionate decreases of the synthesis of the Ssa1 and Ssa2 members of the hsp70 heat-shock gene family, and of the Hsp82 and Hsc82 heat-shock proteins. Quantification of pulse-labelled, immunoprecipitated lacZ fusion proteins showed that synthesis of each of these proteins was disproportionately decreased in prt1-1 mutant cells. Although the mRNAs of affected genes were shown to be polysomal in mutant cells, they were fragmented and of decreased abundance, as indicated by transcript analysis and in vitro translation. Thus the mRNAs of these hsp genes become degraded under the conditions of limited translation initiation that are imposed by the prt1-1 mutation. This untimely mRNA degradation accounts for the disproportionate decreases in polypeptide synthesis in prt1 mutant cells. We propose that sequences at the translation initiation site of SSA2 mRNA bring about the observed mRNA fragmentation.

Alleles↗
PubMed1993

An impaired RNA polymerase II activity in Saccharomyces cerevisiae causes cell-cycle inhibition at START.

Saccharomyces cerevisiae cells harboring the temperature-sensitive mutation rpo21-4, in the gene encoding the largest subunit of RNA polymerase II, were shown to be partially impaired for cell-cycle progress at a permissive temperature, and to become permanently blocked at the cell-cycle regulatory step, START, at a restrictive temperature. The rpo21-4 mutation was lethal in combination with cdc28 mutations in the p34 protein kinase gene required for START. Transcripts of the CLN1 and CLN2 genes, encoding G1-cyclin proteins that, along with p34, are necessary for START, were decreased in abundance by the rpo21-4 mutation at a restrictive temperature. Increased G1-cyclin production, by expression of the CLN1 or CLN2 genes from a heterologous GAL promoter, overcame the rpo21-4-mediated START inhibition, but such mutant cells nevertheless remained unable to proliferate at a restrictive temperature. These findings reveal that START can be particularly sensitive to an impaired RNA polymerase II function, presumably through effects on G1-cyclin expression.

Cell Cycle↗