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J Kurjan

Publications and source records attributed to J Kurjan.

35 records · Page 2Linked to original sources
PubMed1991

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

The carboxyl termini of alpha subunits of mammalian G proteins have been implicated in receptor interactions. We have used a genetic analysis to test such a role for the carboxyl terminus of Scg1, the alpha subunit involved in the yeast pheromone response pathway. A 22-amino-acid truncation (scg1Amb451) resulted in defects in growth and cellular morphology. This phenotype is similar to the null phenotype and represents constitutive activation of the pheromone response pathway; it could result from various effects, including protein instability or constitutive guanine nucleotide exchange, as reported for some altered mammalian G alpha s constructs. A 5-amino-acid truncation (SCG1Och468) resulted in pheromone response and mating defects in both a and alpha cells, which is consistent with defects in interactions with the pheromone receptors. Lysine-to-proline mutations near the carboxyl terminus (SCG1Pro467 and SCG1Pro468) resulted in pheromone response and mating defects, the severity of which differed in a and alpha cells. This differential effect in the two mating types suggests that the specificity for the interactions with the two pheromone receptors may involve different residues of the Scg1 carboxyl terminus. Mutations leading to constitutive activation of the pathway were recessive, whereas mutations that result in decreased pheromone response and mating were partially dominant. These relationships are consistent with the model for the mechanism of action of the G protein subunits in the pheromone response pathway and indicate the importance of the stoichiometry of components of this system.

Base Sequence↗
PubMed1991

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Several domains of guanine nucleotide-binding proteins are conserved and form the guanine nucleotide-binding pocket. Mutations in these domains in EF-Tu, ras, and Gas have been shown to result in informative phenotypes. We made several analogous changes in SCG1, which encodes the alpha subunit of the G protein involved in pheromone response in yeast. The scg1Lys388 and scg1Ala391 mutations resulted in severe growth and cell morphology defects; this phenotype is similar to the null phenotype and results from constitutive activation of the pheromone response pathway. On the basis of the model for the action of the yeast G protein, the effect of these mutations is consistent with the effect of analogous mutations in ras, which result in a transforming phenotype. The SCG1Ala322 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous G alpha s mutation, which results in a defect in stimulation of adenylate cyclase. The scg1Val50 mutation, which is analogous to the transforming mutation rasVal12, resulted in multiple effects, including defects in growth, cell morphology, and mating. Some of our results and interpretations are different from previously published results of others for the same mutation in SCG1; specifically, our gene replacement of this mutation resulted in high basal activation of the pheromone response pathway, consistent with a GTPase defect, which was not seen previously with scg1Val50 on a low-copy plasmid. Implications of these phenotypes are discussed.

Base Sequence↗
PubMed1991

Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway.

The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.

Amino Acid Sequence↗
PubMed1991

The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin.

Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.

Agglutination↗
PubMed1990

Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.

Animals↗
PubMed1989

AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.

Agglutinins↗
PubMed1987

The yeast SCG1 gene: a G alpha-like protein implicated in the a- and alpha-factor response pathway.

We have identified the SCG1 gene by its ability to suppress the pheromone-supersensitive sst2-1 mutation. The nucleotide sequence of SCG1 suggests that it encodes a 54 kd protein homologous to the alpha subunit of the vertebrate G proteins transducin, Gs, Gi, and Go. SCG1 expression and function are haploid-specific; haploid scg1 cells grow into very small colonies consisting of large, abnormally shaped cells, whereas a/alpha scg1/scg1 diploids show wild-type morphology, growth, and sporulation. We postulate that the SCG1 product is involved in the pheromone response pathway, and propose two models for the function of the SCG1 product. Expression of the rat alpha s gene in yeast partially complements both the sst2 and scg1 defects, indicating a high level of conservation of sequence and function between SCG1 and mammalian G alpha subunits.

Amino Acid Sequence↗
PubMed1987

Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone.

Strains of both haploid mating types containing sst2 mutations are altered in response to pheromone; MATa sst2 cells are supersensitive to alpha-factor, and MAT alpha sst2 cells are supersensitive to a-factor. This phenotype suggests that SST2 encodes a component of the pheromone response pathway that is common to both mating types. We have cloned the SST2 gene by isolation of multicopy plasmids that complement the sst2-1 mutation. One such plasmid contained a 4.5-kilobase HindIII fragment that was able to complement the sst2-1 mutation in high or low copy number, integrated at the SST2 locus, and resulted in an sst2 phenotype when disrupted, indicating that this fragment contained the SST2 gene. We identified the functional region of the complementing DNA fragment by transposon mutagenesis. Sequencing of this fragment identified an open reading frame encoding 698 amino acids at a position that correlated well with the functional region. Expression of an Sst2-beta-galactosidase fusion was haploid specific and induced by exposure to pheromone. We discuss a model in which induction of the SST2 product results in inhibition of a component of the pheromone response pathway, resulting in desensitization to pheromone.

Base Sequence↗
PubMed1986

Agglutination and mating activity of the MF alpha 2-encoded alpha-factor analog in Saccharomyces cerevisiae.

The MF alpha 2-encoded Asn-5,Arg-7 alpha-factor-like peptide has been shown shown to have similar activity to Gln-5,Lys-7 alpha-factor in morphogenesis and growth arrest studies (S. Raths, P. Shenbagamurthi, F. Naider, and J. M. Becker, J. Bacteriol. 168:1468-1471, 1986). We tested the Asn-5,Arg-7 peptide in agglutination and mating assays and found that its activity was similar to or slightly less than that of the Gln-5,Lys-7 alpha-factor. The Asn-5,Arg-7 alpha-factor-like peptide is thus the most active analog of the Gln-5,Lys-7 alpha-factor known.

Cell Adhesion↗
PubMed1985

Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating.

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.

Base Sequence↗
PubMed1982

Genetic analysis of the processing of a spliced tRNA.

We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutants examined. These enzymes may recognize only limited regions of the precursor RNA. RNA polymerase III behaves as if able to count the number of Us added before termination; and aberrant termination products in two mutants suggest that the secondary structure of the nascent transcript can be very imortant in eukaryotic transcription termination.

Animals↗
PubMed1982

Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor.

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.

Amino Acid Sequence↗
PubMed1982

Mutations at the Saccharomyces cerevisiae SUP4 tRNA(Tyr) locus: isolation, genetic fine-structure mapping, and correlation with physical structure.

The SUP4 tRNA(Tyr) locus in Saccharomyces cerevisiae has been studied by the isolation and characterization of mutations at the SUP4 gene which result in the loss of suppressor function. Most of the mutations act as single-site mutations, whereas about a third of the mutations are deletions of the entire gene. Two meiotic fine-structure maps of the gene were made. The first mapping technique placed 10 mutations plus the sup4+ anticodon on a map by a measurement of levels of recombination between pairs of mutations. The second map utilized a more qualitative estimate of recombination frequency, allowing 69 mutations and the sup4+ anticodon to be mapped. The maps were compared with the physical structure of the gene for the 34 mutations whose nucleotide alteration has been determined by DNA sequencing (Koski et al., Cell 22:415-425, 1980; Kurjan et al., Cell 20:701-709, 1980). Both maps show a good correlation with the physical structure of the gene, even though certain properties of genetic fine-structure maps, such as marker effects and "map expansion," were seen.

Chromosome Mapping↗
PubMed1981

tRNA synthesis: identification of in vivo precursor tRNAs from parental and mutant yeast strains.

In vivo yeast precursor tRNAs have been identified using a modification of the Northern-hybridization procedure. Two species of pre-tRNA Tyr, 1 species of pre-tRNA Ser2 and 2 species of pre-tRNA Serminor have been found in all yeast strains examined, including parental strains and strains harboring mutations affecting tRNA function. One of the tRNA Tyr strains harboring and one of the pre-tRNA SerUCG are the same size as the unspliced pre-tRNAs which accumulate in the yeast mutant rna1. The in vivo tRNA Tyr precursors detected in these studies also appear similar with the RNA species identified when cloned yeast tRNA Tyr is transcribed and processed by Xenopus oocytes and/or Xenopus extracts. We have also studied the precursor and mature tRNA Tyr species from 22 mutants which contain mutations in the SUP4 tyrosine-inserting suppressor locus. The RNA from 2 mutants mapping at the G52 position showing an aberrantly migrating "mature" tRNA Tyr. Although several of those cloned mutant genes showed transcript products of altered size in in vitro transcription studies (1), we did not detect such altered transcripts in vivo.

Animals↗
PubMed1980

Mutations at the yeast SUP4 tRNATyr locus: DNA sequence changes in mutants lacking suppressor activity.

Yeast strains harboring indepjendent mutations within the SUP4 tyrosine tRNA gene have been selected by virtue of their inactivating effect upon the SUP4-o UAA suppressor. Three fourths of the mutations at SUP4 are point alterations; the rest resemble the deletions described by Rothstein (1979). A meiotic genetic fine structure map of the locus was made by crossing 69 of the mutants in all combinations and testing for the frequency of SUP4-o recombinants. The sequences of SUP4 genes cloned from 32 mutant strains were determined by the dideoxynucleotide terminator method, using as primer a synthetic oligodeoxynucleotide corresponding to a sequence adjoining the SUP4 3' terminus. The positions of the DNA sequence alterations showed good colinearity with the positions of the mutations on the genetic map. One of the 26 mutant sites found by DNA sequencing lies within the intervening sequence. At this site three repeat mutations were found, each changing AT leads to TA. Whereas mutations were generally rather uniformly distributed throughout the tRNATyr coding sequence, none occurred in the DNA sequences flanking the mature tRNATyr sequence or in a 12 nucleotide sequence including the 10 bp which constitute the 3' side of the intervening sequence.

Base Sequence↗
PubMed1980

Mutations of the yeast SUP4 tRNATyr locus: transcription of the mutant genes in vitro.

Twenty-nine different SUP4-o tRNATyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacing a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the T psi CG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.

Animals↗